CHAPTER 1

INTRODUCTION TO IN VITRO TOXICOLOGY AND CELL CULTURE

In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing
methods are employed primarily to identify potentially hazardous chemicals and/or to
confirm the lack of certain toxic properties in the early stages of the development of
potentially useful new substances such as therapeutic drugs, agricultural chemicals, direct
food additives, cosmetics, etc. In vitro models offer a number of ethical and economic
advantages over animal testing. These assays are being used earlier in toxicity testing
pipelines, often to determine risk assessment and/or set up controls that will ultimately spare
animal life. The combination of lower-cost and higher throughput assays can help bring
products to market faster. Additionally, in vitro models give researchers an advantage in
understanding the biological processes involved in a toxic response sooner than if they were
depending on the visual inspection of a live animal. In vitro techniques represent tools that
the toxicologist can utilize at various stages of drug development to design and select
compounds to perform specialized evaluations and to address preclinical or clinical issues
that may arise. The use of in vitro technologies contributes to the scientific quality and
economy of the safety assessment process as well as to the toxicologists' commitment to the
three Rs of reduction, refinement, and replacement, and a fourth R, responsibility.
Advancements in cell culture have given researchers a viable alternative and/or
complement to live animal testing. In vitro investigations in specialized cell cultures have
been used to clarify the mechanism of toxic action of chemicals on a particular target organ.
Cell culture is the technique by which either prokaryotic or eukaryotic cells are grown under
controlled conditions. In practice the term "cell culture" refers to culturing of cells derived
from multicellular eukaryotes, especially animal cells. The cellular systems used in toxicity
studies include primary cells, genetically modified cells, immortalized cells, stem cells, and
cells in different stages of differentiation and transformation, co-cultures of different cell
types, etc. The focus is on the cellular models used to study chemical toxicity, specific
toxicity end points at levels including molecular, and the extrapolation of data obtained from
in vitro models to the context of in vivo. Thus, in vitro toxicology can potentially replace
animal use in toxicological evaluations to a very great extent.
Based on the characteristics of the source of cells, cell cultures are classified as follows:
Primary cell culture: One that is started from tissues/organ freshly removed from the
organism. They are generally prepared by treating the original tissue with cell dispersing
1

agents and planting the cell suspension on a glass/plastic substrate, where the cells adhere and
grow. When cells from primary culture are serially transferred it causes a selection of a single
cell type to become predominant and multiply at a constant rate. The primary cell culture is
then said to have originated a cell strain. Life span is limited to 50 to 60 passages and then it
dies out and the strain comes to an end. During multiplication of a cell strain some cells
become altered and acquire a different morphology, grow faster and have unlimited life. It is
now designated as a cell line.
Established cell lines or continuous cell culture: eg Hep2, ME-180. One in which
the cell nuclei contain chromosome numbers other than the diploid number (heteroploid). The
cells are capable of indefinite replication, not anchorage dependent, not inhibited by density
of population, malignant, and capable of growing in defined media consisting of low
molecular weight nutrients without protein supplements. The transformed cell lines or
modified cell lines are those cells which are either derived from tumor cells or have been
manipulated in some way i.e., transfection with oncogenes or treatment with carcinogens.

1.1 Types of mammalian cultures

Explant cultures: Cell culture initiated from a fragment of tissue/ organ planted in the
medium/plasma clot. The cells migrate out and form a monolayer, and the original tissue

becomes necrotic.
Monolayer cultures: Consists as a layer of cells growing on the surface of a culture vessel.
Limitation here is the surface area available for the cells to adhere.
Suspension Culture: Anchorage-independent cells grow while suspended in fluid medium.
This would yield very large populations of cells.
Micro-carrier cell cultures: Used for large scale propagation of anchorage-dependent cells
that cannot be cultivated in suspension, but attached to the surface of carrier particles.

1.2 Advantages of cell culture

Reduction of animal use

Cytotoxicity and screening of pharmaceutics, cosmetics,

Reagent saving

etc.
Reduced volumes, direct access, lower cost

Physico-chemical environment

Controlled pH, temperature, osmolarity, dissolved gases

Cell line homogeneity

Availability of selective media, cloning

Characterization

Cytology and immuno-staining are easily performed

Preservation

Can be stored in liquid nitrogen

Physiological conditions

Control of hormone and nutrient concentrations

1.3 Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and
many

biotechnology

products

including

enzymes,

hormones,

immunobiologicals

(monoclonal antibodies, interleukins, lymphokines) and anticancer agents.

Diagnosis and therapy
Screening and studies of cell toxicity mechanisms

CHAPTER 2
ANIMAL CELL CULTURE ESSENTIALS AND LAB MAINTENANCE
2.1 Cell culture lab
Any laboratory, in which tissue culture techniques are performed, regardless of the
specific purpose, must contain a number of basic facilities. These usually include the
following:

A general washing area (Preparation area)

Culture area

a) Washing Area
The washing area should contain large sinks, some lead-lined to resist acids and
alkalis, draining boards, and racks, and have access to demineralized water, distilled water,
and double-distilled water. Space for drying ovens or racks, automated dishwashers, acid

baths, pipette washers and driers, and storage cabinets should also be available in the washing
area.
b) Culture Area
The most desirable arrangement is a small dust-free room equipped with an overhead
ultraviolet light and a positive-pressure ventilation unit. The ventilation should be equipped
with a high-efficiency particulate air (HEPA) filter. A 0.3-m HEPA filter
of 99.97-99.99% efficiency works well. All surfaces in the room should
be designed and constructed in such a manner that dust and
microorganisms do not accumulate, and the surfaces can be thoroughly
cleaned and disinfected. A room of such design is particularly useful if
large numbers of cultures are manipulated or large pieces of equipment
are utilized. Bench space for hot plates/stirrers, pH meters, balances,
water baths and media-dispensing equipment should be available. Refrigerators and freezers
for storing stock solutions and chemicals, a microwave or a convection oven, and an
autoclave or domestic pressure cooker for sterilizing media, glassware, and instruments.
2.2 Instrumentation
The specific requirements of a cell culture laboratory depend mainly on the type of
research conducted; for example, the needs of mammalian cell culture laboratory specializing
in cancer research is quite different from that of an insect cell culture laboratory that focuses
on protein expression. However, all cell culture laboratories have the common requirement
of being free from pathogenic microorganisms (i.e., asepsis), and share some of the same
basic equipment that is essential for culturing cells. The basic equipments are as follows:
a) CO2 incubator
Incubators are designed to promote the growth of microorganisms or cells by
maintaining a constant temperature within a narrow range. There are many types of
incubators available that offer an array of features. Advanced features may include carbon
dioxide (CO2) atmosphere, circulating fans, humidity controls, recording thermometers and
alarm systems. A controlled atmosphere is achieved by using a humidifying tray and
controlling the CO2 tension with a CO2-monitoring device, which draws air from the
incubator into a sample chamber, determines the concentration of CO 2, and injects pure CO2
into the incubator to make up any deficiency. Air is circulated around the incubator by natural
convection or by using a fan to keep both the CO 2 level and the temperature uniform. The
landscape of a typical life science laboratory has changed dramatically over the years but the
CO2 incubator continues to be a staple in the research lab. Although the ultimate goal of
4

maintaining cell culture stocks has not changed, the functioning and operation of CO2
incubators has become more accurate, more reliable and more convenient.
b) Laminar flow cabinet
A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully
enclosed bench designed to prevent contamination of semiconductor wafers, biological
samples, or any particle sensitive device. Air is drawn through a HEPA filter and blown in a
very smooth, laminar flow towards the user. The cabinet is usually
made of stainless steel with no gaps or joints where spores might
collect. Laminar flow cabinets may have a UV-C germicidal lamp to
sterilize the shell and contents when not in use. These hoods are
generally available in 4-ft and 6-ft lengths, the latter being somewhat
more convenient in terms of work space. Two people can work side
by side in a 6-ft hood. This is convenient if the experimental protocol requires two people to
work together. Larger hoods also may be necessary if large-scale tissue culture work is to be
done where many large spinners or roller bottles are handled at the same time.
c) Inverted microscope
An inverted microscope is a microscope with its light source and
condenser on the top, above the stage pointing down, while the objectives and
turret are below the stage pointing up. Inverted microscopes are useful for
observing living cells or organisms at the bottom of a large container (e.g., a
tissue culture flask) under more natural conditions than on a glass slide which
is the case with a conventional microscope.

The stage on an inverted

microscope is usually fixed, and focus is adjusted by

moving the objective lens along a vertical axis to bring it closer to or
further from the specimen. The focus mechanism typically has a dual
concentric knob for coarse and fine adjustments. Depending on the size
of the microscope, four to six objective lenses of different
magnifications may be fitted to a rotating turret known as a nosepiece.
These microscopes may also be fitted with accessories for fitting stilland video cameras, fluorescence illumination, confocal scanning and many other
applications.
d) Cryocan
Cryocans are used for cryopreservation, which is a process where cells or whole
tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or
5

196 C (the boiling point of liquid nitrogen). At these low temperatures, any biological
activity, including the biochemical reactions that would lead to cell death, is effectively
stopped. However, when cryoprotectant solutions are not used, the cells being preserved are
often damaged due to freezing during the approach to low temperatures or warming to room
temperature. Phenomena which can cause damage to cells during cryopreservation mainly
occur during the freezing stage, and include solution effects, extracellular ice formation,
dehydration and intracellular ice formation. Many of these effects can be reduced by using
cryoprotectants. Most commonly used cryoprotectants are DMSO and glycerol.

Expanded equipment and additional supplies:

Water bath

Centrifuge

Refrigerator and freezer (-20C)

Cell counter (e.g., Countess Automated Cell Counter or hemocytometer)

Sterilizer (i.e., autoclave)

Confocal microscope

Flow cytometer

Water de-ionizing unit

Fluorescent microscope

Microplate reader

Deep freezer (-80C)

Micropipettes and/or multi-channel pipettes

pH meter

Microgram balance (Electronic balance)

Lab oven

AGE and PAGE apparatus and accessories

Aspiration pump (peristaltic or vacuum)

Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-well plates)

Syringes and needles

Waste containers

2.3 Aseptic technique and good cell culture practice

This is to ensure that all cell culture procedures are performed to a standard that will
prevent contamination from bacteria, fungi and mycoplasma, and cross contamination with
other cell lines.
Sanitize the cabinet using 70% ethanol every time before commencing work.
Sanitize gloves by wiping them in 70% ethanol and allow them to air dry for 30

seconds before commencing work.

Put all materials and equipment into the cabinet prior to starting work after sanitizing

the exterior surfaces with 70% ethanol.

While working, do not contaminate hands or gloves by touching anything outside the
cabinet (especially face and hair). If gloves become contaminated, re-sanitize with

70% ethanol as above before proceeding.

Discard gloves after handling contaminated cultures and at the end of all cell culture

procedures each time.

Equipment in the cabinet or that which will be taken into the cabinet during cell
culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped

with tissue soaked with 70% ethanol prior to use.

Movement within and immediately outside the cabinet must not be rapid. Slow
movement will allow the air within the cabinet to circulate properly.

Speech, sneezing and coughing must be directed away from the cabinet so as not to

disrupt the airflow.

After completing work disinfect all equipment and material before removing from the
cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry

with tissue. Dispose off tissue by autoclaving.

Sanitize the cabinet with 10 30 min UV light before commencing work and after
finishing work. Warning: Plastics will crack and become brittle over time with
repeated exposure to UV light. Only some cabinets have timed UV lights. Ensure they
are not left on for extended periods.

2.4 Fumigation of rooms using formaldehyde

Remove all unnecessary items from the room, including those that must not be exposed to the
gas, e.g., cell cultures, sensitive electrical equipment, etc.
Clean the room with 70% ethanol to minimize the level of microbial contamination

and to allow good gas penetration

Turn off air-handling system (roof HEPA filter system) and close the room as far as

possible and turn on the air conditioner to fan mode.

Fill the container with formalin solution (10ml/m3 of room volume).
Place the container in middle of the room and leave the room.
Lock the door and display safety warning notice.
Leave for 24-72 h.
Afterwards, turn on cabinet and /or air-handling system.
Leave until the level of formaldehyde reaches an acceptable level.
Alternatively ammonia solution can be placed in a bowl and kept in the room as

ammonia absorbs the formalin vapors and neutralize them.

The room may require cleaning to remove residues of paraformaldehyde.

2.5 Washing and sterilization of reusable lab-ware

Soak in chromic acid solution except metals or directly in detergent.
Wash with detergent.
Rinse with tap-water (continuously or with sequential changes).
Rinse with distilled water (two or more times).
Dry in hot air oven.
Sterilize in autoclave.
Store for use (in dedicated cupboards or racks).

CHAPTER 3
PRIMARY CELL CULTURE (LYMPHOCYTE CULTURE)
AND KARYOTYING
The culture of explants taken directly from living organism (e.g., biopsy material) is
known as primary cell culture. The culture consists of mixed population of cell types.
Frequently, some of the cells survive without undergoing proliferation and will, therefore, be
lost in the increasing population of those which are able to multiply in the conditions supplied
in vitro. Cells from explants may sometimes be converted to cell lines by passage. These may
continue to proliferate for a number of cell generations. In some instances the primary cells
are fused with so-called immortal (cancer) cells to produce a hybridoma line. Many of the
explanted cells will survive only for one or a few passages before dying. Apart from study of
the primary cells per se there are a number of other uses for these cells including the use of
primary fibroblasts as feeder layers for the growth of some embryonic stem cell types. This
chapter deals with the primary culture of lymphocytes from human blood and osteoblast cells
from rat calvariae.
3.1 WBC isolation and culture
Outline
Layer whole citrated blood or plasma, depleted in red cells by dextran-accelerated
sedimentation, on top of a dense layer of Hi-sep. After centrifugation, most of the
lymphocytes are found at the interface between the Hi-sep/metrizoate and the plasma.
Requirements

Blood sample in heparin or citrate (concentration determined by collection container

which will already contain citrate or heparin).

Clean centrifuge tubes or universal containers

PBS

Hi-sep

Serum-free medium

Syringe, Pasteur pipettes or pipette

Procedure

Collect the blood sample in citrated or heparinized container and transport to

laboratory.
NOTE: Human blood may be infected with agents such as AIDS virus or hepatitis and
should be handled with great care.

Dilute 1:2 with PBS, and layer 4 ml onto 2 ml Hi-sep. This should be done in a wide,
transparent centrifuge tube with a cap, such as 15-ml sterile conical tube.

Centrifuge the suspension for 20-30 min at 2000 rpm.

Carefully remove the plasma/PBS without disturbing the interface.

Collect the interface with a syringe or Pasteur pipette, and dilute it to 20 ml in serumfree medium.

NOTE: Pasteur pipettes and syringes with sharp needles should not be used with
human blood. Instead, use a 1ml pipetter or a syringe with a blunt canula.

Centrifuge the diluted cell suspension from the interface at 2000 rpm for 10 min.

Discard the supernatant, and re-suspend the pellet in 2 ml of serum-free medium or

PBS. If several washes are required e.g., to remove serum factors, re-suspend the

10

cells in 20-ml of serum-free medium, and centrifuge two or three times more, and
finally re-suspend the pellet in 2 ml of serum-free medium.

Lymphocytes will be concentrated at the interface, along with some platelets and
monocytes. Some granulocytes may be found in the interface although most will be
found mostly in the Hi-sep/metrizoate, in the pellet created in step 3. Monocytes and
residual granulocytes can be removed from the interface fraction by taking advantage
of their adherence to glass (beads or the surface of a flask) or to nylon mesh. Use a
positive sort by MACS or flow cytometry with specific lymphocyte surface markers if
purer preparations are required.

3.2 Harvest of peripheral blood lymphocytes for chromosome preparation

Outline
To obtain metaphase chromosome preparation from peripheral blood following a
modified method of Hungerford (1965) and to stain the preparation.
Requirements
Colchicine
0.01% colchicine is prepared by dissolving 5mg of colchicine in 50ml of double
distilled water.
Hypotonic solution
0.559 g of KCl is dissolved in 100ml of double distilled water and stored in wash
bottles at 37oC.
Giemsas stain
The stock solution of Giemsa is prepared by dissolving 1g of Giemsa powder in 54ml
of glycerol at 56oC for 60 to 90 min using a magnetic stirrer. After cooling to room
temperature, 84ml of methanol is added and left on the magnetic stirrer for one hour. It is
filtered (No.1 Whatman filter paper) and the filtrate is stored in the refrigerator. The working
solution is prepared by adding 2ml of stock Giemsas solution and 2ml of 10% disodium
hydrogen phosphate solution to 46ml of distilled water (pH 6.8).
Procedure
At the end of the incubation period, add 100 l of 0.01% colchicine solution to arrest

the dividing cells at metaphase.

Incubate the cultures further for a period of 1 hr. Transfer the contents of the vial to a

centrifuge tube and spin at 1500 rpm for 5min.

Discard the supernatant and gently tap the cell button. Add 6ml of hypotonic solution

(0.075 M KCl) and mix gently using a cyclomixer.

Place the tube in a water bath at 37 oC for 15 min, then add 1 to 2 ml of cold fixative
(methanol : glacial acetic acid , 3:1) using a vortex and spin at 1500 rpm for 5 min.

11

Discard the supernatant and re-suspend the cells in freshly prepared fixative. Give

another change of fixative and leave the material in the refrigerator overnight.
Suspend the cell button in a small quantity of fresh fixative (after the cells have been
spun down) and prepare a test slide by placing a drop of the cell suspension on a

cooled slide and dry over a slide warmer kept at 40oC.

Examine the slide under a light microscope and check the cell density and the spread

of chromosomes.
Prepare the rest of the slides after making necessary dilutions of the cell suspension.
Stain the slides in a 4% buffered solution of Giemsas for 5 to 7 min, rinse in distilled
water and air-dry. Under low (10x) and high (40x) power objective lenses of the
microscope, observe several cells in metaphase, with chromosomes showing varying

degrees of condensation amidst interphase nuclei.

Observe Giemsas-stained chromosomes of a metaphase cell (under 100x objective)

and count the number of chromosomes.

Each chromosome will be seen to consist of two sister chromatids held together at the

centromere.
Human chromosomes are classified into seven groups (A to G) based on gross
morphology i.e., length and position of centromere.

3.3 G Banding technique using trypsin and Giemsas (GTG)

Giemsa is the most popular stain for chromosome analysis. Conventional or solid
staining method is very useful in studying satellites, dicentric chromosomes, ring
chromosomes, fragments, double minutes, fragile sites, and breaks. However, chromosome
identification is not possible.
Differential staining techniques include the fluorescent and Giemsas staining
methods that expound light and dark bands along the length of chromosomes. These methods
are indispensable in the unequivocal identification of chromosomes. The Giemsa bands
obtained by digesting the chromosomes with the proteolytic enzyme, trypsin, are the most
widely used in clinical laboratories for routine chromosome analysis.
Outline
To band metaphase chromosomes on a given slide employing G-banding technique
using trypsin and Giemsas (GTG) (Seabright, 1971).
Requirements
Trypsin (HiMedia), 4 % buffered Giemsa solution, sodium chloride (SRL).
Trypsin solution
Trypsin
8 mg
0.9% NaCl
50 ml
Protocol

12

Immerse fresh slides (2 to 5 day- old) in trypsin solution in normal saline at room
temperature for 10 to 15 sec.

The time of exposure to trypsin varies with the age of the slide.

Rinse the slides in saline and then in distilled water. Stain in 4% buffered Giemsa
solution for 5 min.

Rinse the slides in distilled water and air-dry.

The chromosomes will be differentially stained along their length.

The banding pattern of each chromosome (shared by homologous chromosomes

including the active and inactive X-chromosomes in female individuals) will be found
to be specific.

3.4 Human karyogram

The word karyogram means a systematized array of the chromosomes prepared
either by drawing, digitized imaging or by photography, with the extension in meaning that
the chromosomes of a single cell can typify the chromosomes of an individual or even a
species. The chromosomes are cut from a photographic print and arranged on a pre-printed
form. With the application of image processing technology in the field of cytogenetics,
computer-assisted karyotyping systems producing digitized images rapidly replace
photographic prints.
The term karyotype means the description of the normal or abnormal,
constitutional or acquired, chromosomal complement of an individual, tissue or cell line,
using the guidelines of standard human chromosome nomenclature.
An idiogram is a diagrammatic representation of the karyotype.
Outline
To prepare a karyotype from the given photograph of a GTG-banded metaphase showing
a normal 46, XY chromosome pattern.
Requirements
Two prints of the metaphase photograph - one is cut apart and the other is kept intact for
orientation and reference or for recheck with the original metaphase on the slide.

13

Protocol
Cut around the chromosomes leaving some margin of background. If overlapping is
present, cut out one chromosome and then cut the other from another print. Arrange the
chromosomes on a format based on their banding patterns according to ISCN (2009).
The characteristic banding pattern of each chromosome is listed below:
Group A
Chromosome 1: The short arm has two broad bands over the proximal half and four
bands over the middle and distal part of the long arm. The secondary constriction at the
proximal long arm stains heavily and is polymorphic.
Chromosome 2: Four to seven bands may be present over the entire long arm and three to
four over the short arm. The centromere is only lightly stained.
Chromosome 3: The centromere and the paracentric region are fairly densely stained. The
distal parts of both arms stain intensely while the central parts of both arms remain light and
appear as a white band.
Group B
Chromosome 4: Generally more evenly and densely stained. Four evenly spaced wide bands
are present over the long arm and one or two over the short arm.
Chromosome 5: A heavily stained broad band covers the middle third of the long arm. It is
separated from a telomeric band by absence of staining over the distal part of the long arm.
Group C
Chromosome 6: The short arm has a wide proximal area of rather faint staining which sets
off a heavily stained distal quarter of the short arms resembling satellites. The paracentric
region of both arms and the entire long arm, except its telomeric region, are heavily stained.
Chromosome 7: Three distinct bands are found over the long arm. The centromere is
stained. The short arm has two to three bands, one being telomeric.
Chromosome 8: A broad band, sometimes split into three to five individual bands, is located
over the middle part of the long arm. Its proximal region stains lightly. The short arm has one
to two bands.
Chromosome 9: The middle and distal part of the long arm have two wide bands which are
sometimes split into two bands each. The secondary constriction at the proximal long arm
remains light with both Giemsas and quinacrine. It is highly polymorphic and stains almost
selectively with the Giemsa-11 technique.
Chromosome 10: The long arm regularly has three bands and the most proximal band stains
most heavily .The short arm has one broad or two narrow bands over the middle part.
Chromosome 11: Two bands are located over the middle part of the long arm. They may be
separated by a narrow negative band. The centromere is stained and separated from a broad
positive band on the short arm by a narrow negative band.
Chromosome 12: The G-pattern is similar to chromosome 11 but the central band is over
the proximal part in the long arm and is wider than in chromosome 11.

14

X chromosome : There are two to four well marked bands over the long arm, the proximal
one being most prominent, and one over the middle part of the short arm. No difference exists
between the X chromosomes in female individuals.
Group D
Chromosome 13: The long arm stains positively over most of its length except for a narrow
telomeric segment. Up to four individual bands may be present.
Chromosome 14: A broad proximally located band, which may be split into three bands, and
a negatively stained band over the distal third characterize the long arm. There is a narrow
distal band of medium intensity at the end of the long arm.
Chromosome 15: The distal half of the long arm is lightly stained. The proximal half of this
arm has two to three bands.
Group E
Chromosome 16: The paracentric secondary constriction at the proximal arm is heavily
stained in contrast to its negative fluorescence. It is highly variable as a polymorphic trait.
There is a lightly stained band over the junction of the distal to the middle third of the long
arm. The short arm generally shows light staining, but one to two bands may be present.
Chromosome17: The entire chromosome takes little stain and remains rather pale. There is
one usually narrow band over the short arm and one over the distal long arm.
Chromosome 18: This is a densely stained chromosome. Two broad bands appear over the
long arm, the proximal one being brighter and broader than the other. A median narrow band
may be present over the short arm.
Group F
Chromosome 19: There is a fairly positively stained area around the centromere.
Chromosome 20: The distal ends of both arms stain positively while the centromere remains
less stained than in chromosome 19.
Group G
Chromosome 21: It is darker and smaller than Chr 22. In particular, the long arm is densely
stained with one to two proximal bands discernible.
Chromosome 22: There is stain mainly around the centromere, with a pale band in the center
of the long arm.
Y chromosome: The distal half of the long arm is the most densely stained region of the
karyotype with quinacrine. It is variable in length and polymorphic. Two bands may be
visible, the proximal one generally being darker. The short arm and the proximal long arm are
usually positively stained.

15

CHAPTER 4
MAINTANENCE AND CULTURE OF ESTABLISHED CELL LINES
The majority of the cells are derived from vertebrates. With the exception of
hematopoietic cell lines and a few others, most are anchorage-dependent and have to be
cultured on a suitable substrate that is specifically treated to allow cell adhesion and
spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for
suspension culture. Cells that are cultured in suspension can be maintained in culture flasks
that are not tissue-culture treated, but as the culture volume to surface area is increased
beyond which adequate gas exchange is hindered (usually 0.2 0.5 mL/cm 2), the medium
requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating
spinner flasks. The difference between adherent and suspension cell cultures and their
advantages/disadvantages are as follows:

Adherent Cell Culture

Suspension Cell Culture

Appropriate for most cell types, including Appropriate for cells adapted to suspension
primary cultures

culture and a few other cell lines that are nonadhesive (e.g., hematopoietic)

Requires periodic passaging, but allows easy Easier to passage, but requires daily cell
visual inspection under inverted microscope

counts and viability determination to follow

growth patterns; culture can be diluted to
stimulate growth

Cells are dissociated enzymatically (e.g., Does not require enzymatic or mechanical
TrypLE Express, trypsin) or mechanically

dissociation

Growth is limited by surface area, which may Growth is limited by concentration of cells in
limit product yields

the medium, which allows easy scale-up

Growth is limited by concentration of cells in Can be maintained in culture vessels that are
the medium, which allows easy scale-up not

tissue-culture

treated,

but

requires

agitation (i.e., shaking or stirring) for

adequate gas exchange
Used for cytology, harvesting products Used for bulk protein production, batch

16

continuously, and many research applications

harvesting, and many research applications

4.1 Cell line Repositories:

The culture may be established from primary cells, or established cell cultures may be
procured from commercial or non-profit suppliers (i.e., cell banks). Reputed suppliers
provide high quality cell lines that are carefully tested for their integrity and to ensure that the
culture is free from contaminants. Regardless of their source, it is to be made sure that all
new cell lines are tested for mycoplasma contamination before one begins to use them. The
mission of cell repositories focuses on the acquisition, authentication, production,
preservation, development and distribution of standard reference microorganisms, and cell
lines for research in the life sciences. The major cell repositories are as follows:
ATCC (American type Culture Collection)
ECACC (European Collection of Animal Cell Cultures)
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen - German
Collection of Microorganisms and Cell Cultures)
JCRB (Japanese Collection of Research Bioresources)
Cell line Repository in India:
The repository at National Centre for Cell Science (NCCS complex, University of
Pune

Campus,

Ganeshkhind,

Pune

411

007,

Maharashtra,

India.

Website:

http://www.nccs.res.in/, Email: patole@nccs.res.in, milindpatole@hotmail.com) is the only

repository that houses human and animal cells in India. The NCCS repository serves to
receive, identify, maintain, store, cultivate and supply animal and human cell lines and
hybridomas. The repository has procured cultures from various sources within the country
and abroad from 35 animal species. A major bulk of the cell lines stocked in the repository
has been procured from the American Type Culture Collection (ATCC) and the European
Collection of Animal Cell Cultures (ECACC).
After receiving cells from organizations such as ATCC or NCCS, the flask in which
the cells are received should be wiped thoroughly with 70% ethanol and the morphology of
the cells should be observed. The cells should also be checked for contamination or any
change in the medium. After clear observation, the cells are kept in CO 2 incubator for one
day without any disturbance. The next day, the medium is removed and the cells are subcultured for further processing.

4.1 Subculture of Adherent (monolayer) Cell Lines

17

Adherent cell lines will grow in vitro until they have covered the surface area
available or the medium is depleted of nutrients. Prior to this point the cell lines should be
sub-cultured in order to prevent the culture dying. To subculture the cells they need to be
brought into suspension. The degree of adhesion varies from cell line to cell line but in
majority of cases proteases, e.g. trypsin, is used to release the cells from the flask.
Eyeball the cells - View cultures using an inverted microscope to assess the degree of

confluency and confirm the absence of bacterial and fungal contaminants.

Check the pH of the culture medium by looking at the color of the indicator, phenol
red. As a culture becomes more acid the indicator shifts from red to yellow-red to
yellow. As the culture becomes more alkaline the color shifts from red to fuchsia (red
with a purple tinge). As a generalization, cells can tolerate slight acidity better than

they can tolerate shifts in pH above pH 7.6.

Cell attachment: Are most of the cells well attached and spread out? Are the floating

cells dividing cells or dying cells which may have an irregular appearance?
Percent confluency: The growth of a culture can be estimated by following it toward
the development of a full cell sheet (confluent culture). By comparing the amount of
space covered by cells with the unoccupied spaces one can estimate percent

confluency.
Cell shape is an important guide: Round cells in a un-crowded culture is not a good
sign unless these happen to be dividing cells. Look for doublets or dividing cells. Get

to know the effect of crowding on cell shape.

Look for giant cells: The number of giant cells will increase as a culture ages or
declines in "well-being." The frequency of giant cells should be relatively low and

constant under uniform culture conditions.

One of the most valuable guides in assessing the success of a "culture split" is the rate
at which the cells in the newly established cultures attach and spread out. Attachment
within an hour or two suggests that the cells have not been traumatized and that the in
vitro environment is not grossly abnormal. Longer attachment times are suggestive of
problems. Nevertheless, good cultures may result even if attachment does not occur

for four hours.

Keep in mind that some cells will show oriented growth patterns under some
circumstances while many transformed cells, because of a lack of contact inhibition
may "pile up" especially when the culture becomes crowded. Get to recognize the
range of cells shapes and growth patterns exhibited by each cell line.

Procedure for sub-culturing of cells:

18

Remove the spent medium.

Wash the cell monolayer with 1-2 ml of PBS without Ca2+/Mg2+ (CMF-PBS).
Pipette trypsin onto the washed cell monolayer using 1ml per 25cm 2 of surface area.

(e.g., 1 ml T25, 3 ml T75). Rotate flask to cover the monolayer with trypsin.
Incubate in hood for 2-10 minutes depending on the cell line. Some cells may need to
be sitting in the incubator. Too long of a period of trypsinisation, the cells will die.

Not enough, you will not transfer enough cells.

Once the cells start to sheet and the media becomes cloudy, move on to the next step.
You may examine the cells using an inverted microscope to ensure that many (~40%)
the cells are detached and floating. It may help to slap or tap the flasks gently to

release any remaining attached cells.

Re-suspend the cells in a small volume of fresh serum-containing medium to
inactivate the trypsin. Disperse the cells (this is a process to disaggregate clumps or
sheets of cells) by running the suspended cells in the medium three to four times with
the tip of the pipette on the bottom corner of the flask. Be careful not to aspirate your

media into the pipette aid. If this happens, the filter must be replaced.
Transfer the required number of cells to a new labeled flask containing pre-warmed

medium.
Key Points

Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all

traces of serum from the culture medium by washing the monolayer of cells with PBS

without Ca2+/Mg2+
Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged
exposure could damage surface receptors.
Trypsin should be neutralized with serum prior to seeding cells into new flasks,
otherwise cells will not attach.

Sub-culture of suspension cells:

Sub-culturing suspension cells is somewhat less complicated than passaging adherent
cells. Because the cells are already suspended in growth medium, there is no need to treat
them enzymatically to detach them from the surface of the culture vessel, and the whole
process is faster and less traumatic for the cells. Replacement of growth medium is not
carried out in suspension cultures; instead, the cells are maintained by feeding them every 2
to 3 days until they reach confluency. This can be done by directly diluting the cells in the
culture flask and continue expanding them, or by withdrawing a portion of the cells from the

19

culture flask and diluting the remaining cells down to a seeding density appropriate for the
cell line.
4.2 Cell counting and viability assay
Hemocytometer cell count (Trypan blue staining)
Thoroughly clean the hemocytometer and its cover slip and wipe both with 70%

alcohol before use.

Place the cover slip centrally over the counting area and across the grooves. Gently
move the cover slip back and forth over the chamber to appropriate position on the

ruler area.
Mix the cell suspension gently and add an aliquot to the trypan blue solution (100l

cell suspension: 100l dye). The dilution will depend on the cell concentration.
Draw a sample into a micro-pipette after mixing thoroughly and the entire tip on the

pipette to rest at the junction between the counting chamber and the cover slip.
Using a light microscope at low power, focus on the counting chamber.
Count the viable and non-viable cells in both halves of the chamber.

Calculations:
Total number of viable cells = A x B x C x 104
Total dead cell count = A x B x D x 104
Total cell count = viable cell count + dead cell count
% Viability = Viable cell count x 100 / Total cell count
Where A = Volume of cells
B = dilution factor in trypan blue
C= mean number of unstained cells
D= mean number of dead or stained cells
104 is the conversion factor for 0.1mm3 to 1 M
4.3 Cryopreservation
If a cell line can be expanded sufficiently, preservation of cells by freezing will allow
secure stocks to be maintained without aging and protect them from problems of
contamination, incubator failure, or medium and serum crises. Ideally, 1 10 6 107 cells
should be frozen in 10 ampoules, but smaller stocks can be used if a surplus is not available.

20

The normal procedure is to freeze a stock of one to three ampoules as soon as surplus cells
are available, then to expand remaining cultures to confirm the identity of the cells and
absence of contamination, and freeze down a seed stock of 1020 ampoules. One ampoule,
thawed from this stock, can then be used to generate a using stock. In many cases, there may
not be sufficient doublings available to expand the stock as much as this, but it is worth
saving some as frozen stock, no matter how little, although survival will tend to decrease
below 1 106 cells/ml and may not be possible below 1 105 cells/ml.
Factors favoring good survival after freezing and thawing are:
(i) High cell density at freezing (1 106 107 cells/ml).
(ii) Presence of a preservative, such as glycerol or dimethyl sulfoxide (DMSO) at 5
10%.
(iii)Slow cooling, 1C/min, down to 70C and then rapid transfer to a liquid nitrogen
freezer.
(iv) Rapid thawing.
(v) Slow dilution, 20-fold, in medium to dilute out the preservative.
(vi) Re-seeding at 2- to 5-fold the normal seeding concentration. For example, if cells
are frozen at 5 106 cells in 1 ml of freezing medium with 10% DMSO and then
thawed and diluted 1:20, the cell concentration will still be 2.5 105 cells/ml at
seeding, higher than the normal seeding concentration for most cell lines, and the
DMSO concentration will be reduced to 0.5%, which most cells will tolerate for 24 h.
(vii) Changing medium the following day (or as soon as all the cells have attached) to
remove the cryoprotectant. Where cells are more sensitive to the cryoprotectant, they
may be centrifuged after slow dilution and re-suspended in fresh medium, but this
step should be avoided if possible as centrifugation itself may be damaging to freshly
thawed cells.
Procedure:

Once cells get 80-90% confluent, the cells are washed with PBS and trypsinized.

1x106-1x107 cells/ml is taken along with 10% serum medium and cryo-protectant such
as dimethyl sulfoxide (DMSO: 5-10%) in a cryovial.

Then the cells are kept for slow cooling, 1C/min, down to -70C and then the vials
are rapidly transferred to a liquid nitrogen freezer.

4.4 Resuscitation of Frozen Cell Lines

21

It is vital to thaw cells correctly in order to maintain the viability of the culture and
enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic
above 4C; therefore, it is essential that cultures are thawed quickly and diluted in culture
medium to minimize the toxic effects.
Remove ampoule from liquid nitrogen and place in a water bath at an appropriate
temperature for your cell line e.g., 37C for mammalian cells. Submerge only the
lower half of the ampoule. Allow to thaw until a small amount of ice remains in the

vial - usually 1-2 minutes.

Wipe the outside of the ampoule with a tissue moistened (not excessively) with 70%

alcohol.
Slowly, drop wise, pipette cells into pre-warmed growth medium to dilute out the

DMSO.
Incubate cells overnight.
Change media the next morning. Removal of DMSO is critical.
Examine cells microscopically (phase contrast) after 24 hours and sub-culture as
necessary.

CHAPTER 5
IN VITRO CYTOTOXICITY ASSAYS
Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells
may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of
cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or
the cells can activate a genetic program of controlled cell death (apoptosis). Cytotoxicity
assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound
libraries. Researchers can either look for cytotoxic compounds, if they are interested in
developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can
screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before
investing in their development as a pharmaceutical. Assessing cell membrane integrity is one
of the most common ways to measure cell viability and cytotoxic effects. Compounds that
have cytotoxic effects often compromise cell membrane integrity. This chapter deals with
various cytotoxic assays.
Plating out of cells for 96-well plate cytotoxic assay

Trypsinize a sub-confluent monolayer culture, and collect the cells in growth medium
containing serum.

22

Centrifuge the suspension to pellet the cells. Re-suspend the cells in growth medium,
and count them.

Dilute the cells depending on the growth rate of the cell line and allowing 10-20 ml of
cell suspension per 96 well plate.

Transfer the cell suspension to a boat and, with a pipette (multichannel pipette is
preferred), add 200 l of the suspension into each well of the central 10 columns of a
96-well plate, starting with column 2 and ending with column 12 and placing 0.5 10
x 103 cells into each well.

Add 200 l of growth medium to the eight wells in columns 1. Column 1 will be used
to blank the plate reader.

Incubate the plates in a humidified atmosphere at 37 C for 24 hrs, such that the cells
are in the exponential phase of growth at the time the drug / toxicant is added.

Drug / toxins addition

Prepare a serial five-fold dilution of the cytotoxic drug / compound in growth
medium to give ten concentrations. This set of concentrations should be chosen
such that the highest concentration kills most of the cells and the lowest kills none
of the cells.

Once the cytotoxicity of a drug is known, a smaller range of

concentrations can be used.

96-well plate map:
1
2
3
Blan
k

Control

T1

10

T2

T3

T4

T5

T6

T7

T8

11
T9

12
T10

23

B = Blank (Media only)

C = Control (Untreated cells)

T1-T10 = Dose range of drug/toxin

Procedure:
Remove the growth medium using pipette. Take care that the tip of the pipette does

not touch the cell sheet.

Add required volume of growth medium to the cell sheet and add medium containing

defined concentrations of the drug.

Follow the same for solvent control.
Add 200 l of the medium free from the drug to the control wells.
Incubate the plates at 37C in 5% CO2 environment for required time points.

5.1 MTT Assay

Principle
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is cleaved by
mitochondrial dehydrogenase of viable cells, yielding a measurable purple formazan product.
This formazan production is proportionate to the viable cell number and inversely
proportional

to

the

degree

of

cytotoxicity.

Formazan

can

be

quantified

spectrophotometrically. This assay measures the antiproliferative effects of cytotoxic drugs.

Procedure
Add 20 l of 5mg/ml of MTT to each well.
Wrap the plates in aluminium foil and incubate in dark for 2 to 4 h at 37C.
Remove the medium and MTT from the wells and dissolve the remaining MTT

formazan crystals by adding 100l of DMSO to all the wells.

Record absorbance in a micro-plate reader at 570nm (measurement) and 630nm
(reference) immediately, since the product is unstable.

24

Determination of IC50
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a
compound in inhibiting biological or biochemical function. This quantitative measure
indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a
given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or
microorganism) by half. In other words, it is the half maximal (50%) inhibitory concentration
(IC) of a substance (50% IC, or IC50). It is commonly used as a measure of antagonist drug
potency in pharmacological research.
Method:
Plot a graph of the absorbance (y-axis) against the concentration of the drug (x-axis).
The IC50 concentration is determined as the drug concentration that is required to

reduce the absorbance to half that of the control.

The absolute value of the absorbance should be plotted so that control values may be
compared, but the data can then be converted to a percentage-inhibition curve, to
normalize a series of curves.

The percentage inhibition is calculated, from the data, using the formula:

Mean OD of untreated cells (control) - Mean OD of treated cells

x 100

Mean OD of untreated cells (control)

The graph can be plotted with percentage of inhibition (y-axis) against the
concentration of drug (x-axis) and IC50 concentration is determined.

25

CHAPTER 6
MORPHOLOGICAL AND OTHER MICROSCOPIC METHODS FOR ASSESSMENT
OF CELL DEATH
There are different modes of cell death caused due to toxicity and this chapter deals
with the various morphological and other microscopic methods for assessment of cell death

Morphological assessment of the cells

6.1 Acridine Orange/Ethidium Bromide (AO/EB) staining:
Principle:
Acridine orange is a vital dye and will stain both live and dead cells. Ethidium
bromide will stain only cells that have lost membrane integrity, i.e., EB will permeate only
cells which have lost membrane integrity. Live cells will appear uniformly green. Early
apoptotic

cells

green and

contain

green

dots in the nuclei

as

will

stain
bright

consequence

of

chromatin

condensation

and

nuclear

fragmentation.

Late

apoptotic cells will

also incorporate ethidium bromide and therefore stain orange but, in contrast to necrotic cells,
the late apoptotic cells will show condensed and often fragmented nuclei. Necrotic cells stain
orange, but have a nuclear morphology resembling that of viable cells, with no condensed
chromatin.

26

Fig: Photomicrographs of AO/EB stained HEp-2

cells incubated for 24 hrs (A) untreated control
cells. (B) toxin treated cells; arrows showing
apoptotic bodies

Stain Preparation
Stock:Acridine Orange - 10 mg/ml in PBS
Ethidium Bromide 10 mg/ml in PBS
Working Solution:AO - 1 l from stock is diluted with 100 l of PBS
EB - 1 l from stock is diluted with 100 l of PBS
AO/EB stain is prepared by mixing the both stains in 1:1 ratio
Procedure
Seeding: Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth

medium containing serum.

Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count

the cells using hemocytometer.

Add 3 ml of growth media and seed around 1,00,000 cells per well in the 6 well plate.

Drug / toxins addition:Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).
Staining:

Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for
4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend

the cells with growth medium.

Mix the cell suspension with the AO/EB stain in 1:1 ratio on a microscopic slide and

cover with glass cover slip.

Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter (450490 nm).

27

1.2 Hoechst 33258 Staining:

Principle:
Hoechst 33258 stains nuclei of both live and dead cells when examined by
fluorescence microscopy.

Cells are scored apoptotic if their nuclei show chromatin

Fig: Hoechst 33258-stained HEp-2 cancer cells for 24h

(A) Control; (B) treated cells. Arrowheads point to the
cells with abnormal nuclei, specially indicating
fragmentation of nuclei/chromatin.
condensation and marginalization of nuclear beading or other apoptotic morphology. Often,

apoptotic nuclei fragment into smaller structures.

Procedure:Seeding: Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth

medium containing serum.

Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count

the cells using hemocytometer.

Add 3 ml of growth media in each well and seed around 1, 00,000 cells per well in the
6 well plate.

Drug / toxins addition:-

28

Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).

Staining:

Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for
4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend

the cells with growth medium.

Mix the cell suspension with the Hoechst 33258 stain in 1:1 ratio on a microscopic

slide and cover with glass cover slip.

Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter (377355 nm).

6.3 JC-1 Staining

Principle
Mitochondrial membrane potential, m, is an important parameter of mitochondrial
function used as an indicator of cell health. JC-1 is a lipophilic, cationic dye that can
selectively enter the mitochondria and reversibly change color from green to red as the
membrane potential increases. In healthy cells with high mitochondrial m, JC-1

spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the
other hand, in apoptotic or unhealthy cells with low m, JC-1 remains in the monomeric
form, which shows only green fluorescence.

29

Stain preparation:
Stock: 5mg/ml
Working solution: 20 l of stock solution dissolved in 1 ml of DMSO
Procedure:Seeding: Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth

medium containing serum.

Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count

the cells using hemocytometer.

Put a cover slip inside each well of the 6 well plate then add 3 ml of growth medium
and seed around 1,00,000 cells per well in the 6 well plate.

Drug / toxins addition:Treat the cells present in each well of the 6 well plate (with cover slip) with IC50
concentration of the cytotoxic drug / compound prepared in growth medium. One of the wells
should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).
Procedure

After the completion of drug treatment period incubate the cells present in the
wells which has glass cover slips with the JC 1 working solution at 37C for 20

minutes.
After 20 min, take out the cover slip with the adhered cells and keep it on the glass
slide inversely.
The directly observe the mitochondrial depolarization patterns of treated and
Fig: Photomicrographs of JC-1 stained SiHa cervical cancer control
cells for 6 h. (A) control cells; (B) treated cells
cells in
a fluorescent microscope fitted with a 377-355 nm filter, at 400x magnification
and then take photographs.
30

6.4 Annexin V Cy3 Assay for Apoptosis

Principle:
The annexins are a group of homologous proteins that bind phospholipids in the
presence of calcium. In living cells phosphatidylserine [PS] is transported to the inner plasma
membrane leaflet by the enzyme Mg-ATP dependent aminophospho-lipid translocase.
However, during the onset of apoptosis, PS is transported to the external leaflet of the plasma
membrane. PS is then available for binding to annexin V and any of its conjugates in the
presence of Ca2+ ions.
Apoptotic cells can be differentiated from necrotic cells in several ways:
Annexin-Cy3.18 (Ann-Cy3) binds to phosphatidylserine present in the outer
leaflet of the plasma membrane of cells starting the apoptotic process. The binding

is observed as red fluorescence.

6-Carboxyfluorescein diacetate (6-CFDA) is used to measure viability. When this
non-fluorescent compound enters living cells, esterases present hydrolyze it,
producing the fluorescent compound, 6-carboxyfluorescein (6-CF). This appears

as green fluorescence.
Cells can be incubated either with Ann-Cy3 or 6-CFDA separately, or with the two
compounds simultaneously. After labeling at room temperature, the cells are
immediately observed by fluorescent microscopy. Live cells will be labeled only
with 6-CF (green), while necrotic cells will be labeled only with Ann-Cy3 (red).
Cells in the early stage of apoptosis, however, will be labeled with both Cy3 (red)
and 6-CF (green).

Seeding: Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth

medium containing serum.

Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count

the cells using hemocytometer.

Add 3 ml of growth media in each well and seed around 1, 00,000 cells per well in the
6 well plate.

Drug / toxins addition:31

Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).
Staining:

Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for
4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend

the cells with growth medium.

Mix the cell suspension with the Annexin stain in 1:1 ratio on a microscopic slide and

cover with glass cover slip.

Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter.

6.5 Comet assay for DNA damage

The comet assay or single cell gel electrophoresis (SCGE) is a rapid, sensitive and
relatively simple method for detecting DNA damage at the level of individual cells.

Reagents
Fig: DNA fragmentation in toxin treated SiHa cervical cancer
cells as revealed in the comet assay. Comet images of DNA
double strand breaks at 12 and 24 h treatment of toxin.

1) 1 % Normal agarose
2) 1 % Low melting agarose
3) Electrophoresis buffer (pH 10)
32

NaOH (AR)
12 g
Na2EDTA
372 mg
Make up the volume to 1 litre with distilled water.
4) Neutralization buffer (pH 7.2)
Dissolve 12.11 g Tris-Base in 250 ml of distilled water.
5) Lysis solution (pH 13 to 14)
NaCl
146.1g
Na2 EDTA
37.2
Tris
1.2 g
Add the above ingredients to 700 ml distilled water and dissolve. Add about 12 g
pelletized NaOH in small quantities and stir until the salts dissolve completely into the
solution. Adjust the pH of the solution to 10.0 using 0.1N HCl or NaOH and make up to 990
ml with distilled water, filter-sterilize and store at room temperature. Add 10 ml of Triton X
-100 (1%) to the above solution and refrigerate for 30 - 60 min prior to use.
Dyes used
Ethidium bromide
Add 5 l ethidium bromide stock (10 mg/ml) per 100 ml gel solution for a final
concentration of 0.5 ug/ml.
Method
Processing of cells
Trypsinize the cells in T25 flask and pellet it. Wash the pellet thrice in PBS.
Electrophoresis apparatus (set up)
The apparatus used for electrophoresis is of the conventional type. However, the gels
used for the assay are different. Since the comet assay involves the microgel electrophoresis
system, gels are loaded in 18 x 18 mm area on a fully frosted microslide. Two samples, each
containing about 1000 -2000 cells, could be conveniently cast on each slide. 8 to 12 such
slides could be placed on the platform of the electrophoresis apparatus as shown in the figure
below.

Reservoir buffer

()

Gel
cells

containing

(+)

Microscope slide

33

Preparation of slides

Drop gently the 200l of 1% normal agarose in PBS at 65C on to a fully frosted
micro-slide, cover immediately with a cover slip and place over a frozen ice pack
for about 5min.

Remove the cover slip after the gel had set. Mix the cell suspension from one
fraction with 1% low melting agarose at 37C in 1:3 ratio.

Apply 100l of this mixture quickly on top of the gel, coated over the micro-slide
and allow to solidify as before.

Give a third coating of 100l of 1% low melting agarose on the gel containing the
cell suspension and allow to solidify.

Similarly, prepare the slides for each cell fraction.

Cell lysis

After solidification of the agarose, remove the cover slips and immerse the slides
in ice-cold lysis solution at 4C for 16h.

Perform all the above operations in low lighting conditions in order to avoid any
additional DNA damage.

Electrophoresis

Place the slides horizontally in an electrophoresis tank after being removing them
from the lysis solution.

Fill the reservoirs with electrophoresis buffer until the slides just immerse in it.

Allow the slides to stand in the buffer for about 20 min (to allow DNA unwinding)
after which carry out the electrophoresis at 0.8v/cm for 15 min.

34

After electrophoresis, remove the slides, wash thrice in neutralization buffer and
gently dab to dry.

Add a few drops of the working solution of ethidium bromide on to the gel and
cover the slide with a cover slip.

Examine the stained DNA in the cells at 200x and 400x magnifications using a
fluorescent microscope equipped with a 365nm excitation filter and a 435nm
barrier filter.

Measure the lengths of DNA migration (comet tail) in these cells using CASP
software. Score about 60-100 comets per point.

Application of CASP software

The comets are analysed using CASP software. The images are used to estimate the
DNA content of individual nuclei and to evaluate the degree of DNA damage representing the
fraction of total DNA in the tail. Cells are assigned to five classes: 0 (<7% of the DNA in the
tail undamaged), 1 (715%), 2 (1522%), 3 (2230%) and 4 (>30%, maximally damaged)
accordingly.

CHAPTER 7
MOLECULAR TECHNIQUES FOR ASSESSING TOXICITY IN VITRO
35

The development of molecular techniques has yielded innovative alternative tools for
understanding and demonstrating the mechanisms underlying various biological functions. It
has also shed considerable light on a number of targeted action mechanisms of various
pharmaceutical, toxicological agents and provides a guide for researchers to study the
molecular basis of the various effects caused by these agents. This chapter deals with a few
molecular techniques for assessing toxicity in vitro.
7.1 Immunocytochemistry (ICC)
a) Peroxidase conjugated Super Sensitive Polymer method for cell cultures
Introduction
ICC is the technique used to detect and localize antigens by means of labeled
antibodies through Ag-Ab interaction that are visualized by enzyme-substrate-chromogen
reaction.
Principle
In these techniques an enzyme-labeled antibody is used to link a cellular antigen
specifically to a chromogen that can be more readily visualized in light microscope.
A lot of methods are available in this technique
i) Direct labeling method
In this method, the enzymes are directly labeled with the primary Ab, so it is a direct
method. This method is more expensive and less in sensitivity.
ii) Indirect labeling method
In this method, the enzymes are labeled with the 2Ab, which was produced against
primary Ab. So it is an indirect method. This method is very cheap and easy to handle.
iii) ABC method
In this method, the 2Ab and avidin are labeled with the biotin and enzyme,
respectively. Avidin has high affinity to biotin. These two biomolecules are used to elongate
the chain and make possible to detect an earlier stage of cancer.
iv) Polymeric method
This technique permits binding of a large number of enzyme molecules to a secondary
antibody via the dextran backbone. The benefits are many, including increased sensitivity,
minimized non-specific background staining and a reduction in the total number of assay
steps as compared to conventional technique.
The simple protocol is
Application of primary Ab
Application of super enhancer
Application of enzyme-labeled polymer secondary Ab
Application of the substrate chromogen.
The result is outstanding sensitivity, signal intensity, low back-ground staining and reduced
non specific binding.
Aim
36

 To detect and localize Bcl-2 in cells by means of super sensitive polymer HRP.
To detect and localize p53 in cells by means of super sensitive polymer HRP.
Required reagents
I) Primary Abs
1. Bcl-2
2. p53
II) Super sensitive polymer HRP kit (Ready to use)
Contents
i) Peroxidase block
ii) Power block
iii) Super enhancer
iv) Super sensitive poly - HRP
v) DAB chromogen
vi) DAB substrate buffer
III) Required materials
1. Distilled water
2. Pressure cooker or micro-oven
3. pH paper or pH meter
4. Moisture chamber
5. Slide carrier
6. Stainless steel trough
7. Electronic weighing balance
8. 30% Hydrogen peroxide
9. Acetone
10. Methanol
Required chemicals
a) Disodium EDTA
b) Trisodium citrate
c) Disodium hydrogen phosphate
d) Potassium dihydrogen phosphate
e) Sodium chloride
f) Tris buffer
g) 1N HCl
h) Hematoxylin
Required solutions to be prepared
10% PLL (100ml; Sigma)
It is mainly for used as an adhesive, to keep the cells intact on the slide during
immunostaining.
Alternatives
2% APES in acetone (100ml; Sigma)
Chrome alum & Gelatin solution
AR solution (for Ag retrieval)
Tris EDTA buffer: pH- 9.0
Tris
- 6.05g
Disodium EDTA
- 0.744g
Distilled water
- 1000ml

37

Alternatives:
Citrate buffer (pH- 6.0)
Trisodium citrate
- 2.94 g
1N HCl
- 5ml
Distilled water
- 1000ml
Modified citrate buffer ( pH- 6.2)
Citric Acid (anhydrous) - 1.92g
Disodium EDTA
- 0.744g
Distilled water
- 1000ml
These solutions are mainly used to unmask the antigen and also to enhance the
reaction between Ag & Ab.
3) Wash Buffers:
Phosphate Buffered Saline (PBS), pH- 7.4
Disodium hydrogen phosphate - 17.5g
Potassium dihydrogen phosphate - 1.5
Sodium chloride
- 17g
Distilled water
- 1000ml
Alternatives :
Tris Buffer Saline (TBS), pH- 7.4-7.6
Tris buffer
- 0.605g
Sodium chloride
- 8g
1N HCl
- 4ml
Distilled water
- 1000ml
These solutions are mainly used to wash the unbound Abs from the cells, and also
used to maintain the pH of the cells.
Procedure
i) Cell cultures are grown onto APES (Amino Propylene Ethoxy Silane) coated slides.
Note:
Do not allow cells to dry at any stage of the staining procedure.
Carry out the steps of incubation with antibody at 37C.
Use appropriate controls for each antibody tested.
ii) Cold acetone
- 15 min Fixation
iii) 0.5% H2O2 in Methanol - 1 min To block endogenous enzyme
iv) Transfer to TBS buffer (pH 7.6) 5 min x 3 times washing
v) Pressure cookerize in TBS buffer (pH 7.6) - 10 min - Heat induced Ag retrieval.
vi) Plunge the pressure cooker into sink with water - 20 min - Cooled to room temperature
vii) Transfer to TBS buffer (pH 7.6) - 5 min x 3 times washing
viii) Power block
10 to 15 min
- To block non-specific reaction
with the other tissue Ag
ix) Drain and cover cells with

To identify Tr markers by Ag Ab reaction

1 hour
5 min x 3 times to wash unbound Abs
To enhance the reaction between
primary and secondary Abs
xi) Super-enhancer
- 30 min
xii) TBS buffer
- 5 min x 3 times to wash unbound Abs
To elongate chain and
also to label the enzyme
xiii) Super-sensitive poly HRP - 30 min
x)
TBS buffer
concerned
primary Ab

38

xiv) TBS buffer

- 5 min x 3 times to wash unbound Abs
xv) Colour development with working
colour development solution
5-8
min
- To give colour to the Ags
xvi) TBS buffer
- 5 min x 3 times
To
wash
xvii) Tap water
5 min
- To wash
xviii) Hematoxylin 1 min
- To counter-stain
xix) Tap water
5 min
- To wash excess stain
xx) Air dry and mount with the mounting medium and view under the microscope.
xxi) Results:
Tr. Markers
Brown
Nucleus
Blue
b) Immunocytochemistry on cytology smears
Required materials
1) Cold acetone
2) 0.5% H2O2 in methanol (only for hemopoietic smears)
3) Tris buffer
4) Primary antibodies
5) Super-sensitive polymer/HRP/DAB kit
Procedure
1) After smear preparation, allow it to air dry for minimum 18h.
2) Fix in cold acetone for 2-3 min.
3) Further fix in 0.5% H2O2 in methanol for 5min ( only for hematopoietic smears)
4) Keep in Tris buffer for 5min x 3 times
5) Power block for 15 min
6) Drain and wipe the surrounding of the smears; incubate with concerned primary
antibody for 1h.
7) Wash with Tris buffer for 5min x 3 times.
8) Incubate with super-enhancer for 30 min.
9) Wash with Tris buffer for 5min x 3 times.
10) Incubate with super-sensitive polymer/HRP for 30 min.
11) Wash with Tris buffer for 5min x 3 times.
12) Develop color with DAB- working solutions for 8-10 min.
13) Wash with Tris buffer for 5min x 3 times.
14) Wash in distilled water for 5 min.
15) Counter-stain with hematoxylin for 1min.
16) Wash in distilled water for 5min.
17) Air dry, clear in xylene, mount with DPX.
Result
Tr. Marker Brown
Nuclei
- Blue
7.2 Gene Expression Studies Adopting Polymerase Chain Reaction (PCR)
Introduction
The starting template for a PCR reaction can be DNA or RNA. DNA is usually the
appropriate template for studying the genome of the cell or tissue (as in inherited genetic
diseases, somatic mutation in a tumor, or somatic rearrangement in lymphocytes) and for the
39

detection of DNA viruses. For information on gene expression in a cell or tissue or the
presence of genomic RNA in a retrovirus such as HIV, RNA is the appropriate template. RNA
can be better than genomic DNA for detecting structural changes in long genes, since
amplifying the spliced RNA transcript instead of the genomic sequence greatly reduces the
length of DNA to be handled without losing any of the coding regions where clinically
significant deletions may be expected.
RT-PCR combines cDNA synthesis from RNA templates with PCR to provide a
rapid, sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify
the expression of mRNA, often from a small concentration of target RNA. The template for
RT-PCR can be total RNA or poly (A) + selected RNA. RT reactions can be primed with
random primers, oligo (dT), or a gene-specific primer (GSP) using a reverse transcriptase.
RT-PCR can be carried out either in two-step or one-step formats. In two-step RTPCR, each step is performed under optimal conditions. cDNA synthesis is performed first in
RT buffer and one tenth of the reaction is removed for PCR. In one-step RT-PCR, reverse
transcription and PCR take place sequentially in a single tube under conditions optimized for
both RT and PCR.
One Step RT-PCR for 25l reaction volume
This protocol serves as a guideline for one-step RT-PCR with a 25l reaction volume.
Reverse transcription and PCR are carried out sequentially in the same tube.

All

components required for both reactions are added during setup, and there is no
need to add

additional components once the reaction

has

been

started.

The

protocol has been optimized for 1 pg 2 g of total RNA. Optimal reaction

conditions, such as incubation times and temperatures during PCR amplification, will
vary and need to be determined individually
Procedure
1.

Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-

PCR Buffer, and RNase-free water, and place them on ice. It is important to mix the
solutions completely before use to avoid localized differences in salt concentration.
2.

Prepare a master mix according to Table 1.

The master mix typically contains all the components required for RT-PCR except

the template RNA. Prepare a volume of master mix 10% greater than that required for
the total number of reactions to be performed. A negative control (without template
40

RNA) should be included in every experiment

3.

Mix the master mix thoroughly, and dispense appropriate volumes into PCR

tubes. Mix gently, for example, by pipetting the master mix up and down a few times.
4.

Add template RNA (2 g/reaction) to the individual PCR tubes.

5.

Program the thermal cycler according to the program described below

6. Start the RT-PCR program while PCR tubes are still on ice. Wait until the
thermal cycler has reached 50C. Then place the PCR tubes in the thermal cycler.

PCR Reaction Mix

Component
volume/reaction
Master Mix
RNase-free water (provided)
variable
5x OneStep RT-PCR Buffer
5.0l
dNTPs mix (containing 10mM of each dNTP)
1.0 l
Primer A
variable
Primer B
variable
5x OneStep RT-PCR enzyme mix
1.0 l
RNase Inhibitor
variable
Template RNA added at step 4
variable
Total volume

25.0 l

Final concentration
--1x
400 M of each dNTP
0.6 M
0.6 M
--5-10 units/reaction
1 pg 2 g/reaction
---

* Contains 12.5 mM MgCl2

A final primer concentration of 0.6 M is optimal for most primertemplate systems.
However, in some

cases

using other primer

concentrations (i.e., 0.51.0M) may

improve amplification performance

RT PCR Reaction conditions

1. Reverse transcription: 30 min 50C. A reverse-transcription reaction temperature
of 50C is recommended. However, if satisfactory results are not obtained using 50C,
the reaction temperature may be increased up to 60C
2. Initial PCR activation: 15 min 95C. Hot start Taq DNA is activated in this step.
Reverse transcriptases are inactivated and the cDNA template is denatured.
3. Step Cycling

41

Denaturation

0.5-1min at 95C.

Annealing

0.5-1min at 50-58C (< 5C below Tm of primers)

Extension

1min 72C

Number of cycles

25-40

Final extension

10min

Analysis
Analyze the PCR reaction products by agarose gel electrophoresis of a 5 l aliquot
from the total reaction. The products should be readily visible by UV trans-illumination of the
ethidium bromide-stained gel.
7.3 Protein Expression Studies Adopting Western Blot
Principle
The Western blot is an analytical technique used to detect specific proteins in a given
sample of tissue homogenate or extract. It uses SDS gel electrophoresis to separate proteins
based on their size. The proteins are then transferred to a membrane (typically nitrocellulose
or PVDF), where they are detected using antibodies specific to the target protein.
Extraction of protein
Reagents
Dignam buffer (pH: 7 to 9) (25 ml)

Hepes 10mM (59.575 mg)

MgCl2 1.5mM (74.25 l from 100 mg/ml stock)

KCl 10mM (186.4 l from 100 mg/ml stock)

DTT 0.5mM (38.56 l from 50 mg/ml stock)

PMSF 0.5mM (43.54 l from 50 mg/ml stock)

NP40 0.1% (25 l)

Store at 40 C
Note: PMSF and NP40 should be added fresh to the lysis buffer.

Procedure

42

The cells are collected and centrifuged.

The supernatant is discarded and dignam lysis buffer is added to the pellet.

The pellet is lysed by passing through a fine syringe and incubated in deep freezer
(3 hours to overnight, freeze-thaw cycle can also be done).

Then it is thawed and centrifuged at 12,000 to 15,000 rpm at 4o C.

The supernatant is collected and stored in deep freezer (-70 o C) for later use.

Estimation of protein concentration (Bradford assay)

Principle
Bradford reagent uses Coomassie blue G-250. Without protein, the solution is redbrown and it is an acidic solution. When protein binds the pKa of the dye shifts causing the
dye to become blue. The dye is measured at 595 nm in a spectrophotometer. Bradford dye is
easy to use, fast and sensitive.
Reagents

Bradford reagent
Dissolve 100 mg of Coomassie blue G250 in 500 ml of ethanol, add 100 ml of
85% of phosphoric acid and make volume to 1 liter with distilled water.

0.1M PBS

Standard protein solution

Dissolve 5 mg of BSA in 50 ml of 0.1M PO 4. This solution contains 100

g/pm/ml
Procedure

Take 0.1 ml of sample solution and make the volume to 1 ml with 0.1 M PBS (pH
7.5).

Pipette appropriate aliquots of BSA containing 0-100 g protein. Make the

volume to 1ml with 0.1 M phosphate buffer (pH 7.5) in all tubes.
43

Add 5 ml of Bradford reagent to all the tubes and mix thoroughly.

Record the absorbance at 595 nm against the reagent blank.

Plot a standard A595 Vs g protein in standard.

Determine the protein content in the sample extract from the standard curve.

Calculate the amount of protein per ml sample.

Poly-Acrylamide Gel Electrophoresis (PAGE) and Western Blot

Reagents
1. Acrylamide/Bis (30% T, 2.67% C)
29.2 g acrylamide and 800 mg NNbis-methylene-acrylamide are dissolved in 100
ml of de-ionized water. Filter and store at 4C in the dark (30 days maximum).
2. 10% (w/v) SDS
10 g SDS is dissolved in 90 ml water with gentle stirring and brought to100 ml with
de-ionized water.
3. 1.5 M Tris HCl (pH 8.8)
18.15 g Tris base is dissolved in 80 ml de-ionized water. Adjust pH to 8.8 with 6 N
HCl and bring total volume to 100 ml with de-ionized water and store at 4C.
4. 0.5 M Tris HCl (pH 6.8)
6 g Tris base is dissolved in 60 ml de-ionized water. Adjust pH to 6.8 with 6 N HCl
and bring total volume to 100 ml with de-ionized water and store at 4C.
5. 10% APS (Fresh daily)
100 mg Ammonium persulfate dissolved in 1 ml of de-ionized water.
6. N N - Tetramethyl ethylene diamine (TEMED)
Commercially available
7. Sample buffer (SDS reducing buffer)
1.25 ml 0.5 M Tris-HCl (pH 6.8), 2.5 ml glycerol, and 2 ml 10% (w/v) SDS are
added to

0.2 ml of 0.5% (w/v) bromophenol blue and brought to total volume of 9.5 ml

with 3.55 ml de-ionized water. The buffer is stored at room temperature. 50 l mercaptoethnaol is added to 950 l of sample buffer prior to use.
8. 10x electrophoresis buffer (pH 8.3)
30.3 g Tris base, 144.0 g glycine and 10 g SDS are dissolved in 800 ml de-ionized
water and volume is brought to 1 L with deionized water and stored at 4C.
Procedure
Gel formulations (10ml)
10 % running gel is prepared by mixing the reagents as shown below.
De-ionized H2O

4.1 ml
30% degased acrylamide/bis

3.3 ml
1.5 M Tris HCl (pH 8.8)

2.5 ml
10 % (w/v) SDS

0.1 ml
44

10% APS
0.1 ml
TEMED
0.004 ml
5 % stacking gel is prepared by mixing the reagents as given below.
De-ionized H2O

5.7 ml
30% degased acrylamide/bis

1.7 ml
0.5 M Tris HCl (pH 6.8)

2.5 ml
10 % (w/v) SDS

0.1 ml
10% APS
0.05 ml
TEMED
0.005 ml
Separation of proteins
Proteins are separated by SDS Polyacrylamide gel electrophoresis based on their
size as described by Laemmli (1970). By heating the sample under denaturing and reducing
conditions, proteins become unfolded and coated with SDS detergent molecule, acquiring a
high net negative charge that is proportional to the length of the polypeptide chain. When
loaded on to a gel matrix and placed in an electric field (100V), the negatively charged
protein molecules migrate towards the positively charged electrode and are separated by a
molecular sieving effect. A molecular weight protein marker (-galactosidase, ovalbumin,
carbonic anhydrase, -lactoglobulin or lysozyme) that produces band of known size is used to
help identify proteins of interest.
Transfer of proteins
After the separation of proteins by SDS-PAGE, the separating gel is rinsed with
distilled water and equilibrated in cold transfer buffer. Transfer sandwich is assembled in the
following order from anode (+) to cathode (-).

Reagents
Towbin buffer (for transfer)
For 250ml
Water

93.75ml

Tris (25mM) 0.76g

Glycine

3.60g

45

Methanol

50ml

SDS

0.094g

pH 8.2-8.4, store at 40 c
TBST (for washing)
For 500ml preparation
Tris

0.6g

Nacl

4.383g

Tweeen 20

0.25ml

pH 7.4
PBST (for washing)
PBS

500ml

Tweeen 20

0.25ml

pH 7.4
Substrate buffer
Tris

151.42mg/25ml

TAB

10mg

H2O2
Protocol
Soak the gel membrane and pads in towbin for 20-30min

Transfer at 25V (constant voltage) for 1hour

Blocking- 2 hrs at room temperature (5%milk powder in TBS)

Add primary antibody (1:3000 dilution) and incubate for 2 hrs at RT/or overnight at 40 c

Wash with PBST (6 times, 5 min each)

Incubate in 20 Ab (1:3000 dilution) in 1hour at room temperature

46

Wash with TBST (6 times, 5 min each)

Add substrate

Add H2O2 (80ml)

The set-up is assembled in the following way:

Positive end

Fiber pad

Filter paper soaked in transfer buffer

NC Membrane

Gel

Filter paper soaked in transfer buffer

Fiber pad

Negative end

The set up is placed in the transfer apparatus filled with cold transfer buffer and
subjected to electric field.
Blocking
After the transfer, the un-reacted sites on the membrane are blocked using the
blocking solution (5% skimmed milk solution or 5% BSA) to reduce the amount of nonspecific binding.
Detection
After blocking, the protein of interest is detected using primary and enzyme linked
secondary antibodies (horseradish peroxidase-conjugated mouse/rabbit secondary antibody).
Analysis
47

After the unbound probes are washed away, the Western blot is ready for detection of
the probes that are labeled and bound to the protein of interest.

48