Multiplexing Phenotype and Function for More Biologically Relevant Insights - 22

More Biologically Relevant Insights | TUTORIAL

that activate T-cells. Cultures were evaluated over time in a multiplexed format by
measuring cell phenotype, T-cell activation markers, cell proliferation, cell viability,
and secreted cytokine analysis.
Optimized Assay Biochemistry
In the T Cell Activation Cell and Cytokine Profiling Kit, as demonstrated in Figure 1,
live immune cells are distinguished from dead cells by staining with a fluorescent
membrane integrity dye that enters only dead cells or those with a compromised
membrane, staining the nucleic DNA by intercalation. Both late apoptotic cells and
necrotic cells with compromised membranes will stain, thereby marking them for
exclusion in further analysis.
Live cells are immunophenotyped by staining with a fluorescent antibody panel to
separate CD3+ T cells, CD3- non-T cells, CD4+ T-helper cells, and CD8+ T-cytotoxic
cells. The T-cell phenotypes are profiled for the expression of three activation
markers, CD69 (early), CD25 (late), and HLA-DR (even later, with strong stimulation).
In addition, the optimized workflow uses two different capture beads to enable
the quantitative measurement, in a sandwich ELISA format, of effector cytokines
secreted by activated T cells, Th1 cytokine IFNγ and multifunctional cytokine TNFα,
in the same assay well.
The assay is optimized to run on the Intellicyt® iQue Screener PLUS, which has a
wide dynamic range without PMT (photomultiplier tube) adjustment, which enables
high resolution cells and multiplexed cytokine-detection beads from the cell/beads
mixture with no need for additional compensation. The Intellicyt® iQue Screener
PLUS provides high-throughput measurements using a flow cytometry detection
engine and patented air-gap delimited sampling technology enables small volume
assays, fast analysis and facilitates plate-based data analytics.
Straightforward Workflow
The straightforward assay workflow is detailed in Figure 2, on the following page.
T cells or immune cells, such as PBMCs, are activated in the culture plates for the
experimentally-designated time frame. Then the cell and supernatant mixture from
each well is transferred into assay plates along with cytokine IFNγ/TNFα capture
beads. After incubating 60 minutes, the plate is centrifuged, the supernatant aspirated, and the cell-bead mixtures are re-suspended in cytokine detection cocktail
containing anti-IFNγ PE (phycoerythrin) and anti-TNFα PE antibodies.
22

| January, 2019

After 60 minutes incubation, a fluorescent antibody panel cocktail with cell viability
dye is added to the assay plate, including antibodies against CD3, CD4, CD8, CD69,
CD25, and HLA-DR. After an additional 60 minutes incubation, the assay plates are
washed once before sample acquisition on the Intellicyt® iQue Screener PLUS using
the violet, blue and red (VBR) laser configuration. T-cell subtypes, three cell surface
activation markers expressed in a temporal specific manner, and two secreted
effector cytokines are measured as the final readouts.
Results
In this tutorial experiment PBMCs were stained with the proliferation and encoder
dye and were stimulated with three different T-cell activators, CD3/28 Dynabeads,
PHA (phytohemagglutinin) and SEB (Staphylococcal enterotoxin B), using a 12-point,
2-fold serial dilution series. On days 1, 3 and 6 after stimulation, 10 µl samples,

Figure 1.

https://www.intellicyt.com/

Multiplexing Phenotype and Function for More Biologically Relevant Insights

Table of Contents for the Digital Edition of Multiplexing Phenotype and Function for More Biologically Relevant Insights