Publications Review

November 26, 2022

Keywords or phrases:
High Throughput Screening, Multiplex Assay, Flow
Cytometry, T Cell Activation, Antibody Screening,
Biologics, Phenotypic Screening, Small Molecule
Screening, B Cell Activation

Advanced Flow Cytometry Applications

for Antibody, Small Molecule and
Phenotypic Screening

Introduction
Multiplexed, multiparameter screening assays are The iQue® advanced high throughput flow cytometry
foundational to the drug discovery process. By collecting platform represents an equally powerful and versatile
more information, more rapidly – whether the focus is alternative, designed to offer advanced assay capabilities
antibodies, small molecules, or phenotypic screening – with the ease-of-use and cost effectiveness sought by
researchers have more confidence in therapeutic research laboratories. Multiplexed, high-throughput
candidates as they progress through the discovery and measurement of cell-specific parameters, protein analysis,
development process. immunophenotyping, functional assessments and profiling,
including antibody screening and immune cell activation,
Flow cytometry is a powerful analytical tool with many deliver breakthrough biological insights with
applications including antibody and small molecule screening unprecedented speed. The patented sampling method
and cell phenotyping. It can be quite complex, however, for allows for the fastest sample acquisition in the industry, and
researchers to integrate into their workflows, limiting direct because sample volumes can be as small as 1-2 µL per well,
access to the technology and necessitating the use of assay reagents are conserved, leaving sample material
centralized core labs, which can slow the pace of discovery. remaining for further downstream characterization studies.
The platform rapidly processes data from multiple assay

Find out more: www.sartorius.com/ique

plates, is compatible with 96- or 384-well configurations This whitepaper summarizes recent peer-reviewed
and offers continuous plate loading via connection with publications describing use of the iQue® advanced flow
automation systems. The integrated iQue Forecyt® software cytometry platform for antibody, phenotypic, and small
accelerates the transition from acquisition to analysis within molecule screening, as well as novel applications such as
minutes, allowing data to be easily visualized and rapidly extracellular vesicles and siRNA delivery system screening.
interpreted without the need for data extrapolation and
export even for complex biological assays.

Antibody Screening
Biologics remain the fastest growing class of therapeutics in The ability to perform primary screens with large scale,
the biopharmaceutical industry. These therapeutics multiplexed assays leads to comprehensive functional
represent a range of approaches including checkpoint profiles on many candidates, increasing the probability of
modulation, chimeric antigen receptor (CAR)-T cell successfully advancing molecules into downstream studies.
therapy, bispecific T cell engagers and neutralizing The same benefits have been applied to the discovery of
antibodies. While each employ different functional tactics neutralizing antibodies targeted against a range of
to engage their respective targets, they all rely on antibody- infectious agents, including SARS-CoV-2.
based binding molecules directed against specific antigens.
Successful development of these antibody-based Wang, et al., provide a detailed description of the
therapeutics requires technologies that enable rapid development of a high-throughput flow cytometry sample
identification and characterization of candidate molecules preparation and readout workflow using the iQue®
with superior target reactivity and optimized functionality. platform.1 The authors describe how the system offers the
advantages of ease of use, miniaturized sample volume, and
With unprecedented versatility and speed, the iQue® robustness for facilitating the discovery of antibody
platform has become the preferred choice for screening therapeutics and advanced modalities such as bispecifics,
and characterizing antibodies during discovery workflows. antibody-drug conjugates (ADCs), and CAR-T approaches,

2
all of which rely on identifying a specific antibody binding cultured Kupffer cells to recapitulate the non-specific
sequence as an initial step. They note that the tedious interactions that lead to ADC clearance in an in vitro
sample and sample volume required for conventional flow setting. As part of the assay, an iQue® system equipped with
cytometry limits the throughput and thus its use as a primary iQue Forecyt® software was used to count and measure
screening method despite its sensitivity and accuracy. ADC binding and uptake into cells by calculating the mean
fluorescent intensity for each treatment condition. The
To fully utilize the advantages offered by flow cytometry on authors note that while further work is needed to establish
the iQue® platform, the authors multiplex multiple cell how broadly this assay can be applied as a predictive tool
lines in one well to simultaneously quantitate on-target for evaluating the impact of drug conjugation on ADC
activity and non-specific activity along with antibody pharmacokinetics, their results suggest the assay may have
concentration. The ability to measure multiple parameters significant utility for development of ADCs.
coupled with speed and increased accuracy provides gains
in productivity and helps accelerate antibody lead Zhang, et al., sought to improve the rate of complete
discovery. These capabilities are critical to cope with the responses in patients with Hodgkin’s lymphoma to CAR-T
increasing volume of antibody discovery programs. therapy by developing next generation anti-CD30 CAR-T
cells with CD28 (28z) costimulatory domains.5 A central
The following peer-reviewed publications describe use of technique in this study was membrane proteome array
the iQue® high-throughput flow cytometry platform to (MPA) specificity testing performed on an iQue® system.
screen antibodies against conventional drug targets such To validate the antigen recognized by the antibody used in
as G-protein coupled receptors (GPCRs) and during the study, the authors performed CD30 scFv screening
development of complex modalities such as bispecifics, using the MPA. The MPA is a platform for profiling the
antibody-drug conjugates (ADCs) and CAR-T therapies. specificity of antibodies and other ligands that target
human membrane proteins and can be used to determine
GPCRs are one of the largest and most diverse membrane antibody target specificity. Plasmids containing cDNA
protein families and more than 30% of all currently clones of 5,344 membrane proteins (representing over 90%
prescribed drugs target these proteins. Sarkar, et al., used of the human membrane proteome, including CD30) were
the iQue® platform as a high-throughput platform to reverse transfected into HEK-293T cells in 384-well cell-
screen antibodies for the ability to modulate the culture plates. Test antibodies were added to MPA matrix
functionality of parathyroid hormone receptor 1 (PTH1R) plates and binding detected using an iQue® high-
which is a member of the GPCR family.2 This screening throughput flow cytometer.
process allows a more thorough characterization of the
signaling behavior of the receptor. Raman, et al., also used the iQue® platform for an MPA to
assess binding of an ADC targeted against
Zhao, et al., used the iQue® platform to characterize neuroblastoma and small-cell lung cancer via binding a
bispecific antibodies designed to enable transferrin conformational epitope.6
receptor (TfR)-mediated delivery across the blood brain
barrier (BBB) in animal models of glioblastoma (GBM).3 The ability to screen antibodies is also essential for
Anti-angiogenesis therapy is an attractive option for development of therapeutics targeting SARS-CoV-2 and
treating GBM due to the high vascular density of these other pathogens. Recent publications describe the use of
tumors. Two of the best-known anti-angiogenic the iQue® platform to accelerate discovery of antivirals.
therapeutics have failed to show significant benefits in
GBM patients, however, in part because of limited brain Pelzer, et al., used the platform to facilitate epitope
penetration due to the BBB. The authors engineered mapping of the protein targeted by a neutralizing
bispecific antibodies by fusing a vascular endothelial monoclonal antibody in development to treat SARS-CoV-2
growth factor (VEGF)-Trap with a TfR-targeting antibody. infection.7 By identifying and targeting a conserved
The iQue® platform was used to quantify the ability of the epitope, the authors believe that the antibody offers
bispecifics to trigger cellular endocytosis which is required therapeutic potential not only against current variants but
for effective transcytosis of the antibodies through TfR. would be expected to maintain efficacy against future
Using mouse endothelial cells, concentration-dependent variants and possibly even novel coronaviruses.
endocytosis of the bispecific antibodies was confirmed.
A study describing development of an ovine anti-
A cell-based assay system to predict the impact that drug body-based therapy against SARS-CoV-2 infection includ-
conjugation technology is likely to have on the ed use of the iQue® platform for antibody-dependent
pharmacokinetics of an ADC antibody has been developed complement deposition (ADCD) assays.8 Plasma was col-
by Meyer, et al.4 The fluorescence-based assay uses lected from sheep immunized with SARS-CoV-2 whole

3
spike protein or a subunit protein once substantial antibody The platform has also been used to accelerate discovery of
titers were generated. Affinity-purified polyclonal antibod- antiviral therapeutics for a range of pathogens. Using Zika
ies to whole spike protein and the S1 and S2 subunits were as a model virus, Gilchuk, et al., developed and
evaluated in vitro for neutralizing activity, antibody-binding, demonstrated an integrated sequence of technologies,
complement fixation, and phagocytosis. including the iQue® platform, designed to enable rapid
response for discovery of antiviral antibodies.10 As an
Cieslewicz, et al., used multiple antigen-presenting system integral part of the workflow, the advanced flow cytometry
(MAPS) technology in which proteins are presented on a platform was used for high-throughput quantitation of
polysaccharide polymer to induce antibody, Th1, Th17 and monoclonal antibodies, competition binding analysis
CD8+ T cell responses, to engineer a novel vaccine and epitope mapping.
targeting SARS-CoV-2.9 IgG subclass, isotype and FcR-
binding profiling was performed using an iQue® platform.

Phenotypic Screening
Historically, most therapeutic antibodies have been isolated bridging of tumor and effector T cells with a functionally
using high throughput target-based screening. But as the relevant CAR activation reporter signal.13 They demon-
number of validated targets becomes limited and the target strated the utility of this enrichment methodology by
space increasingly competitive, Gonzalez-Munoz et al., isolating novel anti-mesothelin CAR-active single-chain
note that alternative strategies, such as phenotypic fragment variable (scFv) candidates. The iQue® platform
screening, are gaining momentum.11 Cell-based phenotypic was used for screening of soluble scFvs for target cell
screening is necessary to support selection of the most binding as well as CAR T cell end-point cytotoxicity and
promising clinical antibody candidates that deliver the activation assays. The authors conclude that for certain
desired biological effect such as cytotoxicity or activation. membrane-bound protein classes, this type of phenotypic
The iQue® platform enables a wide range of these rapid, functional screening may represent an attractive approach
multiplexed assays which offer an indication of in vivo for identifying CAR-active antibody fragments that are not
behavior. generally recovered or prioritized by classical in vitro
antibody screening technologies.
Comacho-Sandoval, et al.,12 developed and validated an
antibody-dependent cell cytotoxicity (ADCC) assay to Bhatta, et al., used the iQue® system for intracellular B cell
test the efficacy and potency of biopharmaceutical signaling analysis and T cell analysis as part of their novel
products using conventional flow cytometry. Based on their high-throughput, combinatorial, phenotypic screening
experience with this assay under a GLP-cGMP approach to screen hundreds of target combinations for
environment, they transferred it to the high-throughput bispecific antibody development.14 The high throughput
iQue® platform in which they were able to evaluate cell flow cytometry signaling assay was used to identify a potent
membrane permeability, caspase activation and bispecific inhibitor of human B cell activation. In addition,
phosphatidyl serine exposure as characteristics of death the authors identified bispecific antibodies capable of
on target cells in the same sample with low volume of activating different T cell subsets for applications in
acquisition. The authors note that these results oncology or infectious diseases.
demonstrated the high throughput technology is suitable
for use in control quality environments and that the Zhao, et al., studied the potential of a leukocyte
automation provided a faster acquisition and analysis of immunoglobulin-like receptor subfamily B member 2
data with precise and accurate results. (LILRB2) blocking antibody for use in the treatment of
Alzheimer’s disease by improving microglial functions.15 The
T cells engineered to express synthetic CARs acquire novel iQue® platform was used in a green fluorescent protein
targeting and activation properties and have delivered reporter assay to discover ligands that bind LILRB2 and
impressive clinical results for some cancers. Targeting and induce inhibitory signals. Their study demonstrated for the
activation are driven by antibody fragments specific for a first time the molecular mechanisms of LILRB2-mediated
tumor antigen. In response to a growing body of evidence inhibition of triggering receptor expressed on myeloid cells
that a classical approach to antibody isolation may not yield 2 (TREM2) signaling in microglia and demonstrated a novel
optimally performing CARs in the in vivo setting, Fierle, et approach of enhancing TREM2-mediated microglia
al., explored the potential of an in vitro phenotypic CAR functions by blocking LILRB2-ligand interactions. The
library discovery approach that associates antibody-driven authors conclude that these data suggest a novel

4
therapeutic strategy for improving microglial functions. binding proteins, between TEM1-β-lactamase and its
The iQue® platform was also used in another study inhibitor protein, such that target binding disrupts the
describing a therapeutic candidate for Alzheimer’s enzyme−inhibitor complex. A therapeutic antibody
disease. Hettman, et al., are developing a humanized (Herceptin), protein biomarker, and plant virus (cow pea
antibody targeting a specific form of post-translationally mosaic virus) were targeted, demonstrating assays for
modified amyloid beta (Aβ) peptides.16 These peptides are therapeutic drug monitoring, health diagnostics, and plant
seen as attractive antibody targets due to their neo- pathogen detection, respectively. During development of
epitope character and propensity to form neurotoxic the assay, an affimer phage display library was screened
oligomeric aggregates. The iQue® was used to perform against biotinylated hCRP with three rounds of panning. A
cellular antibody uptake experiments to ensure primary screen of outputs from panning was performed
preservation of Fcγ-receptor mediated phagocytosis by using the iQue® system.
the antibodies in development.
Three publications highlight the versatility of the iQue®
Pfaff-Kilgore, et al., described the use of functional high-throughput flow cytometer for phenotypic screening
screening to identify sites of vulnerability in the envelope in yeast studies. Arines and Li developed an optimized
proteins (E1, E2) of hepatitis C virus (HCV) which drive protocol to analyze membrane protein degradation in
virus-host membrane fusion.17 The authors analyzed the role yeast using quantitative western blot and flow cytometry.19
of each amino acid in E1E2 function. They expressed 545 The authors used the degradation of Ypq1, a vacuole
alanine mutants of E1E2 in human cells and tested them membrane lysine transporter, to demonstrate the protocol,
against dozens of monoclonal antibodies to understand which can be adapted for other organelle membrane
full-length translation, folding, heterodimer assembly, CD81 proteins and thus offers a new tool to study membrane
binding, viral pseudoparticle incorporation, and infectivity. protein regulation in yeast and other model organisms.
The iQue® platform was used to measure expression of the
HCV E1E2 mutation library transfected into HEK-293T Chen, et al., studied the effect of environmental conditions
cells and incubated with anti-HCV antibody. This step was on the evolution of yeast and used the iQue® platform to
an essential part of a comprehensive analysis of these measure DNA content, and thus the impact on genome
proteins and contributed to development of a proposed size, in response to constant or changing conditions.20 The
mechanism by which individual amino acids contribute to genome size was determined using SYTOX green staining
the folding and function of the E1E2. followed by flow cytometry.

Adamson, et al., used iQue® high-throughput flow The pheromone response pathway of the yeast S. cerevisiae
cytometry in the development of new rapid wash-free is a well-established model for the study of G proteins and
point-of-care and in-field diagnostics that would be broadly mitogen-activated protein kinase (MAPK) cascades.
applicable to a diverse set of multimeric targets.18 They Shellhammer, et al., compared new and established
describe improvements to the approach that combines methods for quantification of pheromone-dependent
target recognition and signal generating elements into an MAPK phosphorylation, transcriptional induction, mating
“active” enzyme-switch that directly transduces target morphogenesis, and gradient tracking.21 The authors used
binding into a signal. Their enzyme−inhibitor switch sensor the iQue® platform for dose-response transcription assays.
was developed by insertion of non-immunoglobulin affimer

Small Molecule Screening

While highly innovative biologics often dominate the Liu, et al., have developed a novel, multiplex immune assay
headlines, small molecule therapeutics remain an essential platform using iQue® high-throughput flow cytometry
modality for treating a wide variety of diseases. In fact, of the technology and advanced computational algorithms for
50 new molecular entities (NMEs) approved in 2021, 36 data analysis.23 The assay simultaneously measures T cell
were small molecules.22 Advances in areas such as dynamics including phenotype, time-dependent
computational drug design, artificial intelligence, and expression of activation markers, secreted effector
screening technologies are enabling pursuit of even the cytokines, and proliferation. A kinase chemogenomic
most challenging drug targets. Using the iQue® platform, library was screened and 25 kinase inhibitors with distinct
small molecules representing diverse chemical space can inhibition profiles on early (CD69) and late (CD25)
be rapidly evaluated via biologically and physiologically activation markers and the cytokines IFNγ and TNFα were
relevant assays. identified. Five kinase inhibitors with dissimilar effects on

5
CD69 and CD25 expression were identified, along with four workflow. The TCA assay may be used as a secondary/
MEK1/2 inhibitors with similar activation profiles. The functional assay to profile drugs including immune-
screening revealed three kinase inhibitors for PKC, IKK2, checkpoint inhibitors, bispecific antibodies, adoptive TIL
and MEK1/2 respectively, all with a phenotypic signature cells, CAR-T cells, and inflammation/autoimmune
similar to ruxolitinib, a Jak1/2 inhibitor used to treat inhibitors. The TCA assay, together with the screening of a
myelofibrosis disease. chemogenomic library, may be used as a primary
screening assay for drug repositioning, phenotypic drug
Figure 1 outlines the use of the iQue® Human T Cell discovery and traditional target-based drug discovery.
Activation Kit (TCA assay) in the immune drug discovery

A. Use TCA as a Secondary | Functional Assay

Immune-checkpoint inhibitors
Bi-specific Ab
T Cell Activation
Adoptive TIL cell therapy
Assay (TCA)
CAR-T cell therapy
Inflammation | autoimmune inhibitors

B. Use TCA as a Primary Screening Assay

Drug Repositioning

Experimental Approach Computational Approach Mixed Approach

T Cell Activation Complex Data Analysis or Machine Learning Algorithm

Assay (TCA)
Compound Compound Preclinical or Clinical FDA-Approved

+
Identification Acquisition Development Drug

Chemogenomic Phenotypic Drug Discovery

Library Drug Candidate
Phenotypic Target Target Lead Selection Preclinical
Ion Moving to
HTS Deconvolution Validation | Optimization Testing
Channel Clinical Trial

Others Kinase
Traditional Target-Based Discovery
Drug Candidate
GPCR Target Target Target-Based Lead Selection Preclinical
Moving to
Identification Validation HTS | Optimization Testing
Clinical Trial

Figure 1
Application of the iQue® Human T Cell Activation Kit (TCA Assay) in the Immune Drug Discovery Workflow

6
Figure 2 provides an example of how screening data are
analyzed by iQue Forecyt® software’s multi-plate analysis
and profile map tools.

A. Criteria B. Plate 1 Plate 2

IFNγ (pg/mL) < = 1,000.00

TNFα (pg/mL) < = 1,000.00

% live cells > = 40.0

CD4+CD69+ as % of CD4+ < = 70.0

C.
CD8+CD69+ as % of CD8+ < = 70.0 100
90 CD4+CD89+ as % of CD4+
CD4+CD89+ as % of CD8+
CD4+CD25+ as % of CD4+ < = 70.0 80
CD4+CD25+ as % of CD4+
70
Percentage (%)

CD4+CD25+ as % of CD8+
60 Normalized IFNγ
CD8+CD25+ as % of CD8+ < = 70.0
50 Normalized TNFα

40
CD4+HLA-DR+ as % of CD4+ < = 30.0
30
20
CD8+HLA-DR+ as % of CD8+ < = 30.0
10
0
Plate 1–H08
Plate 1–B08
Plate 2-B08
Plate 1–C04
Plate 2–E11
Plate 1–D07
Plate 1–D09
Plate 2–G07
Plate 1–D10
Plate 1–E08
Plate 1–B05
Plate 1–G05
Plate 1–E07
Plate 2–G11
Plate 1–G06
Plate 1–B04
Plate 1–A03
Plate 1–A04
Plate 1–A10
Plate 1–B10
Plate 1–C03
Plate 1–F10
Plate 1–E10
Plate 1–B10
Plate 1–D11
Count of CD4+ > = 140.0

Count of CD8+ > = 140.0

Plate ID – Well ID

Figure 2
Thresholds Used to Identify Hits That Decrease the Cytokine Secretion and the Expression
of Eleven Cell Surface Activation Markers
(A). Wells that meet the criteria specified in the iQue Forecyt® Profile Map are shown in teal (B) and ranked (C).

To enable high-throughput drug discovery, Ding, et al., assay development group to fill a technology gap for
compared multiplex high-throughput flow cytometry iQue® screening suspension cells and rare and/or primary cells in
assays with several other commonly used immunoassays, small sample volumes and to replace expensive and/or
including MesoScale Discovery (MSD), Luminex, enzyme- inflexible immunoassays for cytokine profiling. The company
linked immunosorbent assays (ELISA), homogeneous time- now has in place an array of high-throughput flow
resolved fluorescence (HTRF), and AlphaLiSA assays.24 cytometry applications ranging from HTS, multiplex
Study results demonstrated by using a modified profiling, and phenotypic to structure-activity relationship
miniaturized iQue® assay protocol, it was feasible to use only (SAR) screening. In their publication, the authors present
20% of samples and assay reagents, thereby reducing details of several projects demonstrating the impact of
sample consumption and reagent cost while maintaining high-throughput flow cytometry on hit, lead, and novel
assay sensitivity, quality, and reproducibility. target identification in the drug discovery process at
AstraZeneca.
Ding, et al., have described the deployment of high-
throughput flow cytometry at AstraZeneca and practical The AstraZeneca team has also developed an automated
considerations for assay development and screening.25 In 384-well plate iQue® based phenotypic assay for measuring
2014, the company added two iQue® systems to its global the protein expression of FOXP3 and CTLA4 in human
high-throughput screening (HTS) center and one to its regulatory T (Treg) cells.26 The team identified new targets

7
directly regulating FOXP3 (the master regulator of Tregs) and identified compounds that promoted either the
expression and the expression of its downstream genes, such stability or degradation of MYC in a KRAS-mutant
as CTLA4, with the potential to stabilize Treg cell phenotype pancreatic ductal adenocarcinoma (PDAC) cell line.28
and function. Screening a library of 4213 structurally diverse Because stabilization of the MYC oncoprotein by KRAS
compounds allowed them to identify a variety of compounds signaling promotes growth of PDAC a better understanding
regulating FOXP3 and CTLA4 expression. Further evaluation of how this stability is regulated may lead to effective
of these and related small molecules revealed three targets therapies for a very challenging cancer.
as potent positive and negative regulators with potential
implications for establishing novel therapies for autoimmune Kuusanmäki, et al., implemented an iQue® flow cytometry-
diseases and cancer. based approach to simultaneously evaluate the ex vivo
sensitivity of different cell populations in 34 primary acute
Inhibition of the anti-apoptotic machinery of cancer cells is myeloid leukemia (AML) samples to seven drugs and 27
a promising therapeutic approach that has driven the rational drug combinations.29 Data demonstrated that
development of small molecule BH3 mimetics which mimic different cell populations present in AML samples have
BH3 proteins by antagonizing the pro-survival function of distinct sensitivity to targeted therapies.
anti-apoptotic proteins, inducing apoptosis in cancer cells.
To qualify as an authentic BH3 mimetic, several criteria Aggressive natural killer cell (NK cell) leukemia (ANKL) is a
must be met, including the need to function directly on the highly aggressive malignancy with poor prognosis and lack
mitochondria of a cell of known anti-apoptotic dependence of targeted therapies. A recent study by Dufva, et al.,
and directly and selectively inhibit the anti-apoptotic elucidated the molecular pathogenesis of ANKL with a
protein with high-affinity binding. Villalobos-Ortiz, et al., combination of genomic techniques along with high-
developed a comprehensive biochemical toolkit consisting throughput drug sensitivity profiling using the iQue®
of BH3 profiling in parallel with high-throughput viability platform.30 Cell lines derived from NK cell neoplasms and
testing to validate BH3 mimetic candidates. As part of this normal NK cells were characterized to identify
workflow, the iQue® system was used for viability testing.27 therapeutically actionable drivers in malignant NK cells.
Results of the study provide insight into ANKL genetics and
Blake, et al., used an iQue® flow cytometry–based assay to a framework for application of targeted therapies in NK-cell
screen a library of more than 800 protein kinase inhibitors malignancies.

Exosome Screening
Curcumin is a natural low molecular weight polyphenol therapeutic has been hindered due to its low solubility and
derived from turmeric which exhibits potent anti- stability in vivo. Yerneni, et al., used the iQue® platform to
inflammatory, anticancer, and antioxidant activity. Recent assess the membrane integrity of exosomes as part of a
clinical trials have analyzed the pharmacokinetics and study to evaluate the feasibility of extracellular vesicle
effectiveness of curcumin for many types of solid tumors as encapsulation to address the challenges of curcumin
well as multiple myeloma and have proven the molecule’s delivery.31
efficacy and safety. The potential of curcumin as a clinical

Cellular Uptake Screening

Yang, et al., engineered a series of ultra-high affinity Cellular uptake efficiency of PAD-DNA polyplexes was
siRNA binders based on the viral protein p19 and performed using the same approach by Ros, et al.33 poly[3-
developed them into siRNA carriers targeted to the aminopropylmethacrylamide-co-N,N-(dimethylamino)
epidermal growth factor receptor (EGFR).32 The iQue® ethyl acrylate] (PAD) copolymers were used to study the
platform was used to measure the time and effect of changing the charge-shifting potential of a series
concentration-dependent uptake of siRNA into cells. of polycations with constant initial charge density, on
The authors conclude that their study suggests that cytotoxicity and cellular uptake efficiency of in vitro DNA
siRNA-carrier affinity can significantly affect the delivery. Their findings suggest that charge-shifting
intracellular fate of siRNA and may serve as a handle for cationic copolymers are a promising class of synthetic
improving the efficiency of delivery. polymers suitable for in vitro DNA delivery.

8
Summary
Screening is a cornerstone of drug discovery and rapid, of the iQue® high-throughput flow cytometry platform
multiplexed assay technologies that are user-friendly and for antibody, small molecule, and phenotypic screening.
accessible to the research laboratory offer the potential No matter the application, the iQue® platform will
to deliver breakthrough insights into cellular and accelerate and streamline workflows, even for those who
molecular processes. The publications summarized in are not experts in flow cytometry, delivering actionable
this compendium demonstrate the power and versatility results in a decreased timeframe.

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