Contents

4 Novel Tools Advancing Cell Biology Research

Enabling products and technologies help answer new questions
and solve problems.

6 Discovery of Potential Soluble Epoxide

Hydrolase Inhibitors Using a High-Throughput
Screening Assay
sEH inhibitors are promising for treating inflammatory and
cardiovascular conditions.

14 Improved Live Cell Imaging Applications

Novel technology is an effective way for researchers to utilize
live cell imaging in their experiments.

22 Cryopreserved PBMCs Retain Phenotype

and Function
Viable cryopreserved PBMCs offer a convenient alternative to fresh PBMCs.

2
Produced with Support from

Biocompare Tel: 800-637-1277

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South San Francisco, CA 94080 www.biocompare.com
Novel Tools Advancing
Cell Biology Research
Enabling products and technologies help answer new questions and
solve problems.
Cell biology research spans a broad range of areas
including cell structure and function, cell develop-
ment, and cells and disease. There are a multitude
of tools, technologies, and techniques available for
investigating each of these topics, and in this eBook,
we look at some of those resources and how they
are being used to answer new questions, solve per-
plexing problems, and help researchers attain ex-
perimental success.
High-throughput screening
High-throughput technologies have accelerated
the discovery of small molecule drug candidates
by enabling the screening of libraries with millions
of compounds to test their activity against selected
targets. Yet because binding does not always pre-
dict success, other parameters are now being con-
sidered. The most commonly asked question these
days is “which compound can enter the relevant
cell type, bind to the target, and revert the cell to a
healthy phenotype?”.
4
The next article in this eBook hones in on an au-
tomated high-throughput screening platform re-
cently developed to identify small molecule inhib-
itors. This automated HTS assay is shown to be an
efficient way to screen compound libraries against
a specific target. In addition, with this automated
format, multiple assays can be easily modified to
encompass a wide range of target enzymes and
compound library combinations.
Live-cell imaging
Advances in microscopy are helping scientists dis-
cover more than ever before at the cellular and mo-
lecular levels. One important “dimension” in mod-
ern microscopy is time. Being able to observe cell
behavior over a continuous time frame can be use-
ful, for example when observing responses to novel
drug candidates. By contrast, getting a “snapshot”
view at a single endpoint does not always convey
the whole story in terms of cellular response and
kinetics. That’s where live cell imaging comes in.
Live cell imaging tools have evolved significantly
and are becoming more accessible than ever be-
fore. These technologies are not only of great inter-
est to scientists working in the pharmaceutical and
biotech industries, they can be of great value when
applied to drug discovery and development.
The third chapter in this eBook reviews the ef-
fectiveness of live cell imaging for monitoring
3D multicellular models, studying subcellular lo-
calization, and investigating cytotoxic activity of
immune cells.
Cryopreserved peripheral blood
mononuclear cells
Peripheral blood mononuclear cells, or PBMCs, are
peripheral blood cells that have one round nucleus,
including lymphocytes (T cells, B cells, and natural
killer cells), monocytes, and macrophages. They are
used in drug discovery, diagnostic development,
5
cancer research, as well as advanced therapies. In
vitro PBMC studies are crucial in cell biology and
immunology research. PBMCs are also used in in
vivo studies, translational research, as well as dis-
ease modeling and toxicity screening.
Frozen (cryopreserved) PBMCs offer many advan-
tages over fresh PBMCs, enabling researchers to
plan their experiments ahead of time with ready-
to-use cells that can be stored in the freezer until
needed. One concern with frozen cells is the po-
tential loss of viability, phenotype, or function re-
sulting from cryopreservation. In the final chapter
in this eBook, we look at the characterization of
frozen PBMCs as well as a direct comparison of T
cell activation responses through measurement of
expression levels of T cell activation markers. The
data shown suggest that functional, viable cryo-
preserved PBMCs offer a convenient alternative to
fresh PBMCs in cell-based assays and immunolog-
ical studies.
Discovery of Potential Soluble
Epoxide Hydrolase Inhibitors
Using a High-Throughput
Screening Assay
sEH inhibitors are a promising strategy for treating inflammatory
and cardiovascular conditions.

Key Features Introduction

• Soluble epoxide hydrolase (sEH) inhibitors
are a promising strategy for the treatment of
inflammatory and cardiovascular conditions.
• We have developed an automated high-
throughput screening (HTS) platform to
identify small molecule inhibitors that
enables testing of a large number of
compounds in days instead of weeks using
minimal sample volumes.
• We screened all 235 compounds in Cayman’s
Anti-Inflammatory Screening Library and
identified 36 compounds as potential sEH
inhibitors using this approach.
Epoxyeicosatrienoic acids (EETs) are cytochrome
P450 (CYP450) metabolites of arachidonic acid
formed by CYP450 epoxygenases and include four
regioisomers: 5(6)-, 8(9)-, 11(12)-, and 14(15)-EET.1
EETs have been shown to have a wide range of
anti-inflammatory and cardioprotective effects.2
However, EETs are rapidly hydrolyzed by soluble
epoxide hydrolase (sEH) to their respective dihy-
droxyeicosatrienoic acids (DiHETrEs), a conversion
that is coincident with significant loss of their bio-
logical activity. Accordingly, the use of sEH inhib-
itors to increase EET accumulation is a promising
strategy for the treatment of inflammatory and car-
diovascular conditions.

• This HTS platform accelerates drug discovery. Here, a high-throughput screen (HTS) is de-
It can be adapted to many other enzyme scribed utilizing a modified version of Cayman’s
activity assays and performed using Cayman Soluble Epoxide Hydrolase Inhibitor Screening
or client-provided compound libraries. Assay Kit adapted for automation and increased

6
SIMPLE &
EFFECTIVE
ASSAYS
Explore more than 650 robust biochemical
and cell-based assay kits to measure:
· Cell viability
· Receptor activation
· Cellular metabolism
· Mitochondrial function
· Enzyme activity
· Oxidative stress
· Transcription factor activation
· Biomarker levels
www.caymanchem.com/assays

HAVE A CUSTOM PROJECT?

Contact our team of experts for assay design,
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We can build quantitative assays on a variety of analysis
and detection platforms to suit your specific needs.
www.caymanchem.com/bioanalysis
throughput. By automating this assay, data that
would normally take 1-2 weeks to generate and
analyze are obtained over the course of three
days. Additionally, the use of automation drasti-
cally improves reproducibility and reduces both
inter- and intra-assay variability. For this screen,
Cayman’s Anti-Inflammatory Screening Library,
which consists of 235 biologically active and struc-
turally diverse anti-inflammatory compounds,
was evaluated. This screening library was chosen
due to the relevance of sEH as a therapeutic target
for inflammation.
Methods
Compound Library and Controls
An HTS of sEH inhibitors was performed in the
HTS facility at Cayman Chemical. Cayman’s An-
ti-Inflammatory Screening Library contains 235
Figure 1. HTS assay workflow and reaction conditions. The Agilent Bravo automated liquid handler performs all liquid transfer steps in
the HTS assay. The platform is integrated with an Agilent BenchCel 4R microplate handler to enable automated microplate handling to
the BioTek Synergy Neo2 multimode microplate reader.
8
compounds supplied as 10 mM stock solutions in
DMSO in three 96-well Matrix™ tube racks. Com-
pounds were reformatted to a single, mapped 384-
well source plate for HTS. From the source plate,
seven barcode-labeled dilution plates were pro-
duced, each containing vehicle, positive control,
and 10 pt ½ log concentration-response curves for
32 compounds. An additional dilution plate was
prepared as described above with 16 compounds.
DMSO was used as the vehicle (negative) control,
and the sEH inhibitor AUDA was used as the pos-
itive control.
Automated HTS Assays
A fully automated platform consisting of three ro-
botic instruments was used for this HTS assay (Fig-
ure 1). All liquid handling steps were performed
using the Agilent Bravo liquid handler equipped
with a 384 ST pipetting head. Assay and dilution
plates were stored in separate stackers and placed
on the pipetting deck of the Agilent Bravo liquid
handler using the Agilent BenchCel 4R microplate
handler as required. Assay plates were transferred
from the deck of the Agilent Bravo liquid handler
to the stage of a BioTek Synergy Neo2 multimode
microplate reader for data acquisition.
Test compounds were assayed in duplicate in black,
barcode-labeled 384-well non-binding plates. First,
human recombinant sEH was diluted in sEH assay
buffer, and 39.5 µl of this solution was transferred
to each well of the assay plate. Next, 0.5 µl of test
compound was transferred from the dilution plate
to the assay plate. The reaction components were
preincubated for 5 minutes at room temperature.
Enzymatic reactions were initiated by the addi-
tion of 10 µl of the sEH substrate PHOME. Assay
plates were placed immediately on a BioTek Syn-
ergy Neo2 multimode microplate reader set to
25°C using the Agilent BenchCel 4R microplate
handler. Kinetic measurements were obtained ev-
ery 3 minutes for a total of 20 minutes using a 360
nm excitation filter and 460 nm emission filter (360
excitation/460 emission). Following completion,
assay and dilution plates were stored in separate
stackers. Enzyme and substrate stocks, as well as
inhibitor and substrate pipet tips, were refreshed
between plates.
Quality Control
The Z’-factor is a widely used metric to evalu-
ate an assay’s suitability for HTS applications.3 It
measures assay robustness by determining the
separation between the positive and negative
controls. The Z’-factor was calculated using the
following equation:
9
where σp is the standard deviation of the positive
control (AUDA), σn is the standard deviation of the
negative control (DMSO), µp is the mean of the pos-
itive control, and µn is the mean of the negative
control. Assays with Z’-factors between 0.5 and 1
are considered excellent assays.
Data Analysis
Enzymatic rates were determined by calculating
the increase in fluorescence versus time from the
slope of the linear portion of the reaction. Maxi-
mum fluorescence was obtained at 20 minutes.
Percent activity values for test compounds were
calculated based on wells containing the vehi-
cle control (100% activity) and wells containing
the positive control (sEH inhibitor AUDA). IC50 val-
ues were calculated using GraphPad Prism with a
four-parameter non-linear regression curve fit.
Some compounds exhibited background fluores-
cent properties at high concentrations. This was
determined by plotting compound fluorescence at
the start of the assay versus compound concentra-
tion. Data were omitted in instances where com-
pound fluorescence caused significant interference
with the assay or data analysis. Data were retained
in instances where compound background fluo-
rescence caused no significant impact to the sEH
inhibitory rate.
Assay Performance
Representative vehicle and positive control re-
sponses are shown in Figure 2. The average Z’-fac-
tor for all assays was 0.60, indicating robust as-
say performance. We achieved a throughput of
20 compounds/hour using the automated HTS
method described in the Methods section (data
not shown).

Results & Discussion
IC50 values for sEH inhibition obtained in the HTS
assay are shown for all 235 anti-inflammatory
Figure 2. Scatterplot showing the vehicle control (solid circles;
DMSO) and positive control (solid squares; AUDA) samples for
the Z’-factor calculation of a representative 384-well plate in
the sEH inhibitor HTS assay. Each point represents an individu-
al well on a single plate (n = 32 for each control). The solid lines
represent the mean fluorescence rates of the positive and neg-
ative controls. The dashed lines indicate 3 standard deviations
above or below the mean. The Z’-factor calculated for this was
data was 0.60, indicating excellent assay performance.
Figure 3. Scatterplot of IC50 values for test compounds. The baseline value used to compare all compounds was 10 μM (-5 Log (M)). Com-
pounds with IC50 values less than 10 μM were considered potential sEH inhibitors, whereas compounds with IC50 values greater than 10
μM were considered unlikely inhibitors. Compounds circled in red exhibited potent IC50 values and/or clear concentration-response curves.
10
compounds in Figure 3. An IC50 value of 10 µM was
used as the baseline value to compare all com-
pounds. IC50 values below 10 µM were considered
potential sEH inhibitors whereas compounds with
IC50 values above 10 µM were considered unlikely
sEH inhibitors. 10 µM was chosen in order to cap-
ture weaker secondary target activity, as would be
anticipated with compounds with known primary
targets. In total, 36 anti-inflammatory compounds
were discovered with sEH IC50 values below 10 µM
in this HTS.
Six hit compounds that exhibited potent IC50 values
and/or clear concentration-response curves are
shown in Figure 4. The main anti-inflammatory tar-
gets, primary target potency, and sEH IC50 values of
these six hit compounds are summarized in Table 1.
SC-75741, an NF-κB inhibitor, and trans-AUCB, a
known sEH inhibitor, were identified as potent sEH
inhibitors in this HTS assay.4,5 Both compounds in-
hibited sEH with potency that exceeded the range
of concentrations tested (10 nM-10 µM). Hence, IC50
values for these compounds are indicated as <10
nM. Given these potencies, these compounds are
prime candidates for secondary screening.
Figure 4. Concentration-response curves of select hit compounds. All data are plotted as mean ± standard deviation of two technical
replicates. The Z’-factor values for assay plates containing these compounds were >0.5. Compounds marked with a triple asterisk (***)
exhibited background fluorescent properties at high concentrations.

11
 Table 1. Comparison of IC50 values for primary target and sEH of six hit compounds identified in this automated HTS assay.
*Data is shown for IC50 values unless otherwise indicated.
Ciclesonide is a prodrug of the glucocorticoid
receptor agonist desisobutyryl ciclesonide.6 Glu-
cocorticoids are amongst the most well-known
anti-inflammatory compounds. Despite cicle-
sonide displaying background fluorescence at
high concentrations, we were able to determine
that ciclesonide has an IC50 value of 100 nM for
the inhibition of sEH. Ciclesonide has limited
solubility in aqueous solutions. The higher com-
pound concentrations used in this HTS assay
may have approached the limit of its aqueous
solubility, which could have contributed to high
background fluorescence.
SCH 79797, an antagonist of proteinase-activated
receptor 1 (PAR1), was found to have sEH inhibitory
activity in this screen (IC50 = 372 nM).7 SCH 79797
inhibits PAR1 activation on vascular smooth mus-
cle cells and endothelial cells as well as α-throm-
bin-induced platelet aggregation. Whether or not
the sEH inhibitory activity of SCH 79797 contrib-
utes in part to these cardioprotective effects could
be explored in follow-up studies.
DuP-697, a selective COX-2 inhibitor, was discov-
ered to be a potential sEH inhibitor (IC50 = 813
nM).8 This finding is especially intriguing given the
extensive role of COX-2 in inflammation. COX-2 is
primarily responsible for the production of pros-
12
taglandins (PGs) from arachidonic acid, a family of
lipid signaling molecules with multiple inflamma-
tory functions. Using dual inhibitors of COX-2 and
sEH, potentially like DuP-697, to halt PG production
concurrent with increasing tissue EET accumula-
tion is an intriguing approach for the treatment of
inflammatory conditions.
LCK Inhibitor inhibits the Src-family non-receptor
protein tyrosine kinase LCK.9,10 LCK is expressed by
T cells and mediates IL-2 production, a hallmark of
T cell activation, in response to T cell receptor sig-
naling. Here, we discovered that LCK Inhibitor also
inhibits sEH activity (IC50 = 6,070 nM). Thus, it could
be of interest for future studies to determine if LCK
Inhibitor increases EET levels and if this is a viable
strategy to dampen aberrant T cell activity.
Conclusions
We have established an automated HTS platform
to identify small molecule inhibitors. This auto-
mated HTS assay is an efficient way to screen com-
pound libraries against a specific target. Given the
throughput, this assay could easily be applied to a
larger compound library to increase the probabil-
ity for hits. With this automated format, multiple
assays can be easily modified to encompass a wide
range of target enzymes and compound library
combinations. This automated HTS assay is appli-
cable to basic research as well as drug discovery.
References
1. Lai, J. and Chen, C. The role of epoxyeicosatrienoic
acids in cardiac remodeling. Front. Physiol. 12, 642470
(2021).
2. Spector, A.A. and Kim, H.-Y. Cytochrome P450
epoxygenase pathway of polyunsaturated fatty acid
metabolism. Biochim. Biophys. Acta 1851(4), 356- 365
(2015).
3. Zhang, J.-H., Chung, T.D.Y., and Oldenburg, K.R. A
simple statistical parameter for use in evaluation and
validation of high throughput screening assays. J.
Biomol. Screen. 4(2), 67-73 (1999).
4. Leban, J., Baierl, M., Mies, J., et al. A novel class of
potent NF-κB signaling inhibitors. Bioorg. Med. Chem.
Lett. 17(21), 5858-5862 (2007).
5. Hwang, S.H., Wagner, K.M., Morisseau, C., et al.
Synthesis and structure-activity relationship studies
of urea-containing pyrazoles as dual inhibitors of
cyclooxygenase-2 and soluble epoxide hydrolase. J.
Med. Chem. 54(8), 3037-3050 (2011).
13
6. Belvisi, M.G., Bundschuh, D.S., Stoeck, M., et al.
Preclinical profile of ciclesonide, a novel corticosteroid
for the treatment of asthma. J. Pharmacol. Exp. Ther.
314(2), 568-574 (2005).
7. Ahn, H.S., Foster, C., Boykow, G., et al. Inhibition of
cellular action of thrombin by N3-cyclopropyl-7-{[4-
(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f ]
quinazoline-1,3-diamine (SCH 79797), a nonpeptide
thrombin receptor antagonist. Biochem. Pharmacol.
60(10), 1425-1434 (2000).
8. Kargman, S., Wong, E., Greig, G.M., et al. Mechanism
of selective inhibition of human prostaglandin G/H
synthase-1 and -2 in intact cells. Biochem. Pharmacol.
52(7), 1113-1125 (1996).
9. Burchat, A.F., Calderwood, D.J., Hirst, G.C., et al.
Pyrrolo[2,3-d]pyrimidines containing an extended
5-substituent as potent and selective inhibitors of lck
II. Bioorg. Med. Chem. Lett. 10(19), 2171-2174 (2000).
10. Arnold, L.D., Calderwood, D.J., Dixon, R.W., et al.
Pyrrolo[2,3-d]pyrimidines containing an extended
5-substituent as potent and selective inhibitors of lck
I. Bioorg. Med. Chem. Lett. 10(19), 2167-2170 (2000).
11. Cayman Chemical Company. Unpublished internal
company data. (2000).
Additional Resource
High-Throughput Screening
Improved Live Cell
Imaging Applications
Novel technology is an effective way for researchers to utilize live cell
imaging in their experiments.
Introduction
A live cell imaging method that continuously cap-
tures the images of cells over time has recently
been applied in various research and development
settings. The purpose of this study was to check the
effectiveness of the live cell imaging system on the
following applications.
• Monitoring 3D multicellular model
• Subcellular localization
• Cytotoxic activity of immune cell
Topic 1. Monitoring 3D multicellular
model: Spheroid formation and screening
3D (Three-dimensional) culture is one of the cell
culture methods that could overcome the limita-
tions of 2D culture by realizing an environment sim-
ilar to an actual tissue. 3D culture without scaffold
lets cells form aggregation by simply increasing
cell-to-cell contact. A spheroid, the simplest struc-
ture formed by scaffold-free 3D culture, mimics a
solid tumor in vivo and exhibits similar properties
in terms of growth kinetics and metabolic rate.1,2
In this experiment, we tried to confirm the an-
ti-cancer effect of nocodazole on the spheroid.
14
To determine the cell type and number suitable
for spheroid formation, two types of cells at vari-
ous concentrations prepared by a FACSCOPE B cell
counter were seeded in a round bottom 96 well
plate to generate spheroids and then they were
observed using Celloger® Mini Plus.
As a result of comparing two types of cells, MDA-
MB-231, a human breast cancer cell line, and Hela
cell, a human cervical cancer cell line, the area oc-
cupied by spheroids was very different in the two
cells despite the same number of cells forming the
spheroids. Hela cells occupied a smaller area and ef-
fectively generated spheroids, expecting to be more
strongly aggregated than MDA-MB-231 (Figure 1).
When spheroids are formed by aggregation of
cells themselves, the degree of aggregation varies
depending on the cell type, number of cells, and
culture method; thereby affecting the spheroid for-
mation.2 If the changes in the spheroid area can be
determined through real-time imaging, we could
know when the spheroid formation is completed
(the time when there are no more changes in area),
and select cells that aggregate more efficiently,
such as Hela cells. (Figure 2).
As a result of checking the spheroid area through
time-dependent imaging after treatment with
Figure 1. Seeding concentration-dependent spheroid formation in two cell lines. Image captured by Celloger® Mini Plus (4X optics) when
MDA-MB-231 (upper) and Hela cells (lower) were seeded with 10,000, 5,000, and 1,000 cells per well in a round bottom 96-well plate and
then cultured for 26 hours.

nocodazole, an anti-cancer drug, it was found that
the spheroid size continued to increase regardless
of drug treatment until 18 hours. Spheroids not
treated with nocodazole continued to increase
even after 18 hours, while in the case of nocodazole
treatment, it was found that it gradually decreased
after 18 hours (Figure 3). Identifying the time when
the effect of drug appears is possible only through
real-time imaging. In the case of nocodazole treat-
ment, the effect of the drug appeared after 18
hours, which is presumably because it takes time
for apoptosis to begin after undergoing abnormal
cell division. More details on apoptosis by noco-
dazole will be introduced in Topic 2.

Figure 2. Dynamics of cell coverage during spheroid formation. Both MDA-MB-231 and Hela cells, seeded at 5,000 cells per well, were
captured every 15min by Celloger® Mini plus (4X optics) and the area covered by a spheroid was estimated using Celloger® Mini Plus anal-
ysis app. A. Cell images showing the analyzed cell coverage in yellow. Images were captured immediately and 25 hours after seeding.
B. Changes in the cell coverage (average, N=3) during spheroid formation.

16
Topic 2. Subcellular localization:
Observing the travel of nucleus in mitotic
arrested by an anti-cancer agent
To observe changes in intracellular structure and
protein movement, it is usually necessary to ex-
press a target protein fused with a fluorescent pro-
tein. We conducted transfection with the plasmid
containing the corresponding gene. Cells stably ex-
pressed green fluorescent protein fused histone 2B
(H2B-GFP), which visualizes the dynamics of chro-
mosomal structure during cell cycle progression.3
We monitored real-time cells to observe nuclear
changes that could predict cell cycle. As shown in
Figure 4, the chromosomes are loosely distributed
throughout the interphase stage (Figure 4A, 0 min),
Figure 3. Increase in spheroid area (average, N=3. Error
bar=standard error) via time. The lower left image is a
spheroid at 18 hours after nocodazole treatment outlined
with red line. The image on the right is a spheroid at 26
hours, and the area reduced compared to the image at 18
hours is marked in yellow. Images used for analysis were
captured every 30min by Celloger® Mini Plus (4X optics).
17
and as they undergo prophase stage, they gradu-
ally are condensed and the fluorescence becomes
more intense compared to interphase (Figure 4A,
after 45 min). After passing through metaphase, in
which the chromosomes are aligned in the center
of the cell (Figure 4A, 75 min~90 min), they are sep-
arated into two sister chromatids, and two daugh-
ter cells are finally generated in telophase (Figure
4A, after 105 min). On the other hand, after treat-
ment with nocodazole,4 which is known to induce
cell cycle arrest, metaphase was formed later (de-
layed) than the ones not treated with nocodazole
(Figure 4B, 165 min). The nucleus in metaphase did
not divide into two and became untangled state
(Figure 4B, 255 min~270 min), and DNA eventually
fragmented resulting in apoptosis (Figure 4B, 1170
min). In the process of cell division, chromosomes
bind to the mitotic spindle, a structure composed
of microtubules, and are pulled to both ends to be
divided. In the case of nocodazole, it is known to
disturb microtubule dynamics in cell division.4 In
addition, it is possible to observe the form of chro-
mosomes condensed in metaphase, anaphase, and
telophase during cell division without fluorescence
labeling through high-resolution bright-field im-
aging of the Celloger (Figure 5).
Topic 3. Cytotoxic activity of immune cell:
NK-killing assay
Natural killer (NK) cells play a critical role in prima-
ry defense by recognizing pathogens, virus-infect-
ed cells, or tumor cells as target and killing them.
Because of this role, they are in the limelight in
cancer immunotherapy as a specialized immune
effector cell that responds to abnormal cells.5 We
performed an assay to evaluate the killing ability
of NK cells through Celloger® Mini Plus imaging
and used NK 92 cell, a lymphoblast derived from
blood as an effector NK cells. K562 cells, a cell line
derived from human chronic myelogenous leu-
Figure 4. Timelapse images of H2B-GFP transfected Hela cells with or without nocodazole . A. Control cells were not treated with nocodazole.
B. Cells were treated with 62.5 nM nocodazole. In each panel, upper images are brightfield images and lower images are fluorescence images.

kemia, were used as a target cell and visualized

through cytoplasmic staining with CellTracker™
Green CMFDA Dye. When the coculture was car-
ried out by increasing the ratio of effector NK92
while fixing the target K562 concentration, it was
confirmed through real-time imaging that the
higher the NK cell ratio, the more efficient the
target K562 apoptosis was induced, thereby re-
ducing the number of fluorescent cells (Figure 6).
Since the receptor of NK cells recognizes and bind
to the target antigen, the NK cells are observed
Figure 5. Image of Hela cells during mitosis taken by
as aggregated with the target cells. It is presumed
Celloger Nano (4X optics)
that the higher the concentration of NK cells, the

18
larger the aggregated size and the higher the immune cells such as T cells and NK cells, measures
probability of killing the tumor cells.6 the amount of 51Cr released when target cells die.
Chromium release assay, a well-known and com- This is difficult to screen over time because there
monly used method for quantifying cytotoxicity by is not only a risk of radiation exposure, but the su-

Figure6.Extinctioneffectofk562cellsdependingontheconcentrationofNK-92cellsNK92cells(effector)wereincreasedinproportiontoK562
cells (target) stained with CellTracker™ Green CMFDA, and cocultured. Bright field and green fluorescence channel images were captured
with Celloger® Mini Plus (4X optics) every hour for up to 20 hours. A. Bright field image tile merged with green fluorescence channel images.
B. Green fluorescence image tile.

19
pernatant containing 51Cr must also be collected it includes a stitching function that can obtain an
and measured repeatedly.7 On the other hand, the image of a larger area (Figure 7).
real-time imaging using Celloger makes it easy to
observe that fluorescence-stained target cells de- References
crease due to apoptosis, thereby allowing quan-
tification of cytotoxic activity of immune cells. In 1. Li, Grace N., et al. Tissue Engineering, 2007, 13.5:
addition, it can increase measurement accuracy as 1035-1047.

2. Han, Se Jik; KWON, Sangwoo; KIM, Kyung Sook.

Cancer Cell International, 2021, 21.1: 1-19.

3. Kanda, Teru; Sullivan, Kevin F.; WAHL, Geoffrey M.

Current Biology, 1998, 8.7: 377-385.

4. Jordan, Mary Ann; Thrower, Douglas; Wilson, Leslie.

Journal of Cell Science, 1992, 102.3: 401-416.

5. Vivier, Eric, et al. Science, 2011, 331.6013: 44-49.

Figure 7. Screen for stitching with Celloger® Mini Plus anal- 6. Prager, Isabel; Watzl, Carsten. Journal of Leukocyte
ysis app after imaging one well with 3X3 tile in Celloger® Biology, 2019, 105.6: 1319-1329
Mini Plus
7. Karimi, Mobin A., et al. PloS One, 2014, 9.2: e89357.

20
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Cryopreserved PBMCs Retain
Phenotype and Function
Viable cryopreserved PBMCs offer a convenient alternative
to fresh PBMCs.
Frozen (cryopreserved) peripheral blood mono-
nuclear cells (PBMCs) offer many advantages over
fresh PBMCs, enabling researchers to plan their
experiments ahead of time with ready-to-use cells
that can be stored in the freezer until needed. One
concern with frozen cells is the potential loss of vi-
ability, phenotype, or function resulting from cryo-
preservation. We report here the characterization
of frozen PBMCs from BioIVT as well as direct com-
parison of T cell activation responses through mea-
surement of expression levels of well-established
T cell activation markers (CD25 and CD69). Data
suggest these frozen PBMCs maintained viabili-
ty and phenotype during cryopreservation. While
stunted, T cells exhibited appropriate activation
responses upon anti-CD3 and anti-CD28 antibody
stimulation. Functional, viable cryopreserved PB-
MCs offer a convenient alternative to fresh PBMCs
in cell-based assays and immunological studies.
Frozen PMBC characterization
Frozen PBMCs were first assessed for viability us-
ing propidium iodide staining and characterized
by flow cytometry using fluorescent antibodies
against the following protein markers:
22
• CD14 (monocyte marker)
• CD56 (NK cell marker)
• CD4 (T helper subset)
• CD8 (T cell subset)
• CD19 (B cells)
Cryopreserved PBMCs from three individual
healthy donors were used for all experiments. Rep-
resentative data are shown in Figure 1. Viability
and composition results are summarized in Table 1.
Average cell viability was determined to be 91.8%
(Figure 1A). The phenotypic composition of each
cell population, expressed as a percentage of the
gated cell population (see Figure 1B), aligned with
expected values. Results suggest these frozen PB-
MCs maintained viability and phenotype during
cryopreservation and thawing.
PBMCs and T cell activation
Peripheral blood mononuclear cells consist of lym-
phocytes (T cells, B cells, NK cells) and monocytes.
These cells give selective responses to the immune
system and are key players in immunity. T cells are
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 Table 1. Viability and composition of frozen PBMCs. All cell
populations were selected by gating on lymphocytes and monocytes
by size and CD45+ expression as shown in Figure 1B. T cells were
further selected using a combined gate of CD4+ and/or CD8+. Frozen
PBMCs from three individual healthy donors were used. Results
indicate the average value and standard deviation as determined by
flow cytometry.
Averaged result
Viability 91.8% ± 2.2%
Population composition
Monocytes 32.0% ± 8.8%
NK cells 4.4% ± 2.0%
B cells 10.1% ± 1.6%
T cells (CD4 subset) 32.9% ± 4.1%
T cells (CD8 subset) 18.0% ± 4.0%

Figure 2. Activated T cells display increased CD25 expression

Figure 1.A) Frozen PBMC viability as determined by
and transiently increased CD69 expression.
propidium iodide staining and flow cytometry. Calculat-
ed cell viability (averaged from three samples) was 91.8%.
B) Phenotypic analysis of frozen PBMCs. CD45+ cells were
analyzed for CD14, CD19, and CD8/CD4 expression to de-
CD69 cell surface markers can therefore be used as
termine the composition of monocytes, B cells, and T cells measures of T cell activation and function.
respectively. For NK cells, CD56 staining was used. Tabu-
lated results are shown in Table 1. To determine whether these frozen PBMCs re-
tained their function, we used flow cytometry to
measure changes in CD25 and CD69 following
activated through extracellular signals that are me-
stimulation with anti-CD3 and anti-CD28 antibod-
diated mainly by T cell receptor (TCR) complexes.
ies. Experiments were performed using both fresh
It has been shown that certain agonists such as and frozen PBMCs.
antibodies to CD3 and CD28 can trigger TCR sig-
naling observable through changes in cell surface CD25 expression
marker expression similar to actual activation of
TCR via antigenic stimulation. Activation of T cells PBMC samples were processed in three separate
results in increased CD25 expression and transient- experiments. For each experiment, cell surface
ly increased CD69 expression (Figure 2). CD25 and marker expression was compared using two sam-

24
ples of fresh cells from different donors and two
samples of frozen cells from different donors. Cells
were activated using RPMI(+) culture media con-
taining anti-CD3 (3 µg/mL) and anti-CD28 (1 µg/
mL) antibodies. RPMI(+) media without activation
antibodies was used as a baseline control. Cells
were stained using fluorescently labeled anti-CD25
antibody at the indicated time points post-activa-
tion and analyzed the same day by flow cytometry
(Figure 3). For analysis, cells were gated by size for
the lymphocyte population using forward scatter
area and side scatter area. Cells were then select-
ed by gating on lymphocytes using CD45+ expres-
sion and gating on T cells using a combined gate
of CD4+ and/or CD8+. The resulting cells were con-
sidered T cells of either CD4+ or CD8+ lineage.
As expected, both fresh and frozen PBMCs showed
an increase in T cell CD25 expression following ac-
Figure 3. Expression of CD25 at 24 hours (A) or 72 hours
(B) post-activation, with donor matched no-activation
control samples for each timepoint. A-B) Representative
histograms, with cells shown gated on CD45+ WBCs and
either CD4+ or CD8+ for T cells. C) Change in percent of
cell population expressing CD25, calculated as the differ-
ence between donor match activation and no-activation
samples (fresh PBMCs, n = 2; frozen PBMCs, n = 4). D) Table
depicting the values graphed in C.
25
tivation with anti-CD3 and anti-CD28 antibodies.
Fresh cells exhibited a larger initial increase in CD25
expression compared to frozen cells post-activa-
tion, though a robust response was still observed
with frozen cells on Days 1 and 3. Longer incuba-
tions resulted in a larger increase in CD25 expres-
sion for both fresh and frozen cells.
CD69 expression
Experiments and gating strategy were run as de-
scribed for CD25, using fluorescently labeled an-
ti-CD69 antibody instead of fluorescent anti-CD25
antibody. Results are shown in Figure 4. As ex-
pected, both fresh and frozen PBMCs showed an
increase in T cell CD69 expression following acti-
vation with anti-CD3 and anti-CD28 antibodies.
Figure 4. Expression of CD69 at 24 hours (A) or 72 hours (B)
post-activation, with donor matched no-activation control sam-
ples for each timepoint. A-B) Representative histograms, with
cells shown gated on CD45+ WBCs and either CD4+ or CD8+ for
T cells. C) Change in percent of cell population expressing CD69,
calculated as the difference between donor match activation
and no-activation samples (fresh PBMCs, n = 2; frozen PBMCs, n
= 4). D) Table depicting the values graphed in C.
Fresh cells again exhibited a larger initial increase type, and function after cryopreservation, offering
in CD69 expression compared to frozen cells a convenient, ready-to-use alternative to fresh cells
post-activation, though a detectable response for most applications.
was still observed with frozen PBMCs. For CD69,
a significantly larger response was observed one Materials and methods
day post-activation compared to three days post
activation for both fresh and frozen cells. This Materials
aligns with the expected transient response of the
• Frozen PBMCs from BioIVT
CD69 cell surface marker.
(#HUMANPBMC-0001993)

Frozen PBMC function summary • Fresh PBMCs (AllCells®)

Functional results are summarized in Figure 5. Pat- • Mouse anti-human CD3, monoclonal
terns of changes in CD25 and CD69 marker expres- (#SAB4700041)
sion matched expected responses for both fresh • Mouse anti-human CD28, monoclonal
and frozen PBMC samples. Frozen PBMCs showed (Calbiochem® #217669-100UG)
weaker initial activation compared to fresh PBMCs
as measured by changes in CD25 and CD69 mark- • Anti-CD25-FITC (#SAB47000363)
er expression. Despite this difference, detectable
• Anti-CD69-FITC (BD Biosciences #555530)
responses were observed with frozen cells at both
24 hours and 72 hours post-activation. These data • Anti-CD14-FITC (#FCMAB181F)
suggest these frozen PBMCs retain viability, pheno-
• Anti-CD56-APC (#SAB4700450)

• Anti-CD4-APC (#MABF416)

• Anti-CD8-PE (#MABF1680)

• Anti-CD19-FITC (#FCMAB184F)

• Propidium iodide (#537060)

• RPMI(+) medium: RPMI containing 10% FBS,

1% Pen/Strep, 1% L-Glutamine

• Phosphate buffered saline (PBS)

Figure 5. Summary of data comparing activation of fresh • FACS buffer: 95% PBS and 5% RPMI(+)
and frozen PBMCs as measured by changes in CD25 and
CD69 marker expression. Though a weaker response was
Cell thawing and treatment
observed with frozen cells, the data suggest these cryopre-
1. Thaw frozen vials of PBMCs in the 37 °C
served PBMCs retain their function.
water bath for 2-5 minutes

26
 2. Add RPMI(+) medium to both frozen and
fresh PBMCs
3. Pellet cells at 300 x g for 5 minutes
4. Remove supernatant
5. Resuspend cells in RPMI(+) medium
6. Count cells on hemocytometer and plate
100,000 live cells in a 200 µL volume
per well into a 96-well plate. Note that
samples will be resuspended in RPMI(+)
media containing anti-CD3 (3 µg/mL final
concentration) and anti-CD28 (1 µg/mL
final concentration) for stimulation and
activation response, or only RPMI(+) media
alone for non-activated samples.
Cell staining and gating on
flow cytometer
1. For all wash steps, use FACS buffer.
2. Begin with samples in a 96-well plate
as described in the “Cell thawing and
treatment” section. Transfer the whole
volume of each well (~200 µL) into individual
microcentrifuge tubes.
3. Add 1 mL FACS buffer to each
microcentrifuge tube.
27
4. Spin down the microcentrifuge tubes in a
centrifuge at 300 x g for 5 minutes. Make
sure cells formulate a pellet. If no visible
pellet, repeat spin of cells.
5. Remove supernatant to 50 µL line on
microcentrifuge tube. Resuspend cells with
light pipetting.
6. Add in the fluorophore-labeled antibody
against the desired cell surface marker and
incubate in the dark for 40 minutes at room
temperature.
7. Repeat steps 3-5.
8. Resuspend cells in 250 µL of FACS buffer
and mix by gently pipetting. Stain cells for
viability if desired.
9. Transfer the full volume (~300 µL) from
each microcentrifuge tube into a 96-well
U bottom plate for analysis using a Guava®
easyCyte™ 8HT Flow Cytometer.
View our full listing of normal PBMCs, disease state
PBMCs, and immune cell subsets from BioIVT. For
more information about our PMBCs and Immune
Cell Subsets, please visit www.sigmaaldrich.com/
bioivt.
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business of Merck KGaA, Darmstadt, Germany.