Agilent BioTek Cytation 7 User Manual

Application Note

Immuno-oncology

Stimulation of Human Peripheral
Blood Mononuclear Cells

Using the Agilent BioTek Cytation 7 Cell Imaging
Multimode Reader to Image and Analyze ELISpot
Assays

Author

Paul Held, PhD
Agilent Technologies, Inc.

Abstract

Human peripheral blood mononuclear cells (PBMCs) are routinely isolated from
blood samples and then used in several fields of research including autoimmune
disorders, infectious diseases, vaccine development and cancers. The ELISpot
Assay monitors ex vivo cellular immune responses to antigenic stimuli. Here we
use the Agilent BioTek Cytation 7 cell imaging multimode reader in conjunction with
Agilent BioTek Gen5 microplate reader and imager software to quantitate changes in

cytokine secretion in PBMCs using the colorimetric ELISpot assayformat.

Introduction

Human peripheral blood mononuclear cells (PBMCs) are
differentially stimulated to secrete a number of cytokines as a
result of a receptor mediated cascade based on the cell type
and the stimuli. The response of this diverse group of cells to
different stimuli offers insights into their role in disease and
the development of treatment modalities.

PBMCs are peripheral blood cells that have a round nucleus.1
These cells consist of lymphocytes (T-, B-, and NK-cells) as
well as monocytes. Other peripheral blood cells either have
no nuclei (erythrocytes and platelets) or have multi-lobed
nuclei (neutrophils, basophils, and eosinophils). In humans,
lymphocytes make up the majority of the PBMC population,
followed by monocytes, and only a small percentage of
dendritic cells.

Cytokines are small molecular weight proteins or peptides
secreted by many cell types (particularly immune system
cells) that regulate the duration and intensity of the immune
response. The cytokine interleukin 2 (IL-2) is a pleiotropic
cellular regulatory molecule that is produced by lymphoid
cells in response to several stimuli. It plays a role in preventing
autoimmune diseases by promoting differentiation of
immature T cells into regulatory T cells.3 In addition, IL-2
causes the differentiation of T cells into effector T cells and
memory T cells when the original T cell was stimulated by
an antigen.4 Interferon gamma (IFN-γ), is a cytokine critical

for innate and adaptive immunity against infections. IFN-γ

is produced predominantly by natural killer (NK) and natural
killer T (NKT) cells as part of the innate immune response,
and by cytotoxic T lymphocyte (CTL) effector T cells once
antigen-specific immunity develops.5 The importance of IFN-γ
in the immune system stems in part from its ability to inhibit
viral replication directly, and from its immune-stimulatory

and immunomodulatory effects. Aberrant IFN-γ expression

is associated with a number of auto-inflammatory and
autoimmune diseases.

T-cell activation is normally initiated by the interaction of a
cell surface receptor to its specific ligand molecule along with
a costimulatory molecule.6 This binding event triggers the
rapid hydrolysis of inositol phospholipids to diacylglycerol and
inositol phosphates by phospholipase C (PLC).

Diacylglycerol is an allosteric activator of protein kinase C
(PKC). PKC activation and inositol phosphates, which trigger
Ca2+ release and mobilization, result in a cascade of additional
cellular responses mediating T cell activation (Figure 1). Two

2

of these cellular responses are the production and secretion

of IL-2 and INF-γ. Triptolide is a diterpene triepoxide that is a

potent immunosuppressant and anti-inflammatory (Figure 2).
Triptolide has been shown to inhibit the expression of IL-2 in
activated T cells at the level of purine-box/nuclear factor and

NF-κB mediated transcriptionactivation.

Figure 1. Schematic of signal cascade for stimulation of IL-2 and INF-γ

secretion.

Figure 2. Structure of triptolide.

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2

While some PBMCs are known to produce IL-2 and INF-γ,

under normal growth conditions little is produced. Only after
stimulation will substantial amounts of the cytokines be
expressed.8 Phytohemagglutinin (PHA) is a lectin that binds
to the sugars on glycosylated surface proteins, including

the Tcell receptor (TCR), and nonspecifically binds them.

The result is the low level stimulation of the signal cascade

required for IL-2 or INF-γ secretion.9 Likewise, Phorbol

myristate acetate (PMA) is a small organic compound, which
has a structure analogous to diacylglycerol, that diffuses
through the cell membrane into the cytoplasm where it
directly activates Protein Kinase C (PKC). When used in
combination with ionomycin, a calcium ionophore, which
triggers calcium release, it results in a moderate level of
cytokine release. However, when PMA and a costimulator,
such as PHA, stimulate PBMC cells concurrently, cytokine
production is strongly enhanced.

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The ELISpot assay procedure is very similar to that of a
conventional ELISA. The plates are first coated with the
appropriate capture antibody. Cultured secreting cells are
added to the wells along with any interested experimental
mitogen or antigen. Cells are maintained for a period of time
after which they are removed. The secreted analyte remains
bound to the capture antibodies in close proximity to the
location on the plate where the cell that produced the analyte
was situated. After removal of the cells and any unbound
materials, a detection antibody (usually biotinylated) is added
followed by an enzyme conjugate with an incubation to allow
binding and a wash to remove unbound materials after each
step. As the substrate is converted by the conjugate enzyme
to colored compounds, spots on the plate membrane bottom
at the locations of the original analyte capture are formed.
The resultant spots are then analyzed/counted using image
analysis. (Figure 3).

Figure 3. ELISpot stain procedure.

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Experimental

Materials and methods

Human IL-2 ELISpot colorimetric kit was obtained from
U-CyTech biosciences (Utrecht, The Netherlands) and a

two color human IFN-γ/IL-2 ELISpot kit was from Cellular

Technology Limited (Cleveland, OH). Phorbol 12-myristate
(PMA), and triptolide (part number T3652) were purchased
from Millipore-Sigma. Ionomycin (part number 407952) was
from EMD-Millipore. Human PBMCs were obtained from
Astarte Biologicals (Bothell, WA). White PVDP membrane
96-well (part number MSIP4W10) were from Millipore-Sigma.

Cell culture: Purified human PBMCs were received and
maintained frozen until needed. After rapid thawing cells
were immediately diluted 1:10 in RPMI-1640 plus 10%
FBS supplemented with 2 mM glutamine, penicillin and
streptomycin. Cells were centrifuged at 300 g for 10 minutes
and the supernatant removed. Cells were resuspended in 10
mL of fresh RPMI media, counted and diluted as needed to
provide a density of 5 × 104 cells/well.

Plate coating: Either a human IL-2 ELISpot kit from U-CyTech

Biosciences or a 2-color human INF-γ/IL-2 kit from CTL were

used for these experiments. PVDF membrane plates are first
coated with the appropriate concentration of capture antibody

(anti-IL-2 or anti-FTN-γ) and allowed to absorb overnight

at 4 °C. The unbound antibody is aspirated and the plate is
manually washed 3x with PBS. The wells are then filled with
a blocking solution (200 µL) and allowed to incubate for at
least 1 hour at room temperature. Blocking buffer is aspirated
without washing immediately before the addition of cells.

Cell seeding: Unless otherwise indicated, cells were plated in
96-well membrane plates previously coated with antibody at
a density of 5 × 104/well. PBMCs were stimulated to secrete
IL-2 with a PMA (50 ng/mL), ionomycin (1 µg/mL) mixture.
Typical experiments used a volume of 100 µL for cells
followed by the addition of 100 µL of stimulant mixture at a

2xconcentration.

Triptolide inhibition: PBMCs were plated at 5 × 104/well
in 50 µL volume of complete RPMI media. After allowing
cells to recover for 1 hour at 37 °C, in a humidified 5% CO2
environment, triptolide treatment was added in complete
RPMI media at 4x of final concentration to each well in 50 µL.
IL-2 stimuli mixture (2x) was then added in 100 µL for a final
volume of 200 µL.

One-color ELISpot assay: The assays were performed
according to the U-Cytech BioSciences kit instructions.

After seeding, cells were incubated for 24 hours, at 37°C in

a humidified 5% CO2 environment plates and then assayed
using an ELISpot kit. Briefly, cells were removed by washing
5x with 250 µL PBS-Tween 0.05% using an Agilent BioTek
MultiFlo FX multimode dispenser. A biotinylated detection
antibody (100 µL) is added to the well and allowed to incubate
for 60 minutes at 37 °C or overnight at 5 °C, after which
unbound detection antibody was removed by washing. A
streptavidin-HRP conjugate was then added (100 µL) and
incubated at 37 °C for 60 minutes. Again, unbound conjugate
is removed by washing. Next a two-part AEC substrate was
added that deposits dye onto the well membrane bottom.
Reactions were halted after 30 minutes at RT by washing with
deionized water (250 µL) 3x using the MultiFlo FX and allowed
to dry in the dark. Entire wells were then imaged.

Two-color ELISpot development: The assays were performed
according to the C.T. L. Immunospot 2-color ELISpot kit
instructions. After seeding, cells were incubated for 24 hours,
at 37 °C in a humidified 5% CO2 environment plates were
then assayed using an ELISpot kit. Briefly, cells removed by
washing 5x with 250 µL PBS-Tween 0.05% using a MultiFlo
FX multimode dispenser. A detection antibody solution

(80µL/well) was added to the well and allowed to incubate

at room temperature (RT) for 120 minutes, after which
unbound detection antibody is removed by washing. Tertiary
solution (80 µL/well) was added and allowed to incubate

for 60minutes at RT. Unreacted reagents were removed by

washing 2x with PBS-Tween, followed by 2 washes with dH2O
and then allowed to air dry in the dark. Blue developer solution

was then added (80 µL/well) and incubated for 15minutes

at RT. Reaction was stopped by washing 3x with dH2O.
Red developer solution was then added (80 µL/well) and
incubated at RT for 7 minutes. Plate was the washed 3x with
dH2O. Plate is air dried in the dark for at least 2 hours prior

toimaging.

Plate washing: Plates were washed according to the assay
kit instructions using a MultiFlo FX. Wash buffer consisted of
PBS (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 7.4
mM) supplemented with 0.05% Tween 20. Unless specifically
indicated, plates were washed five times with 250 µL buffer
per well.

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