Multiplexing Phenotype and Function for More Biologically Relevant Insights - 15

More Biologically Relevant Insights | APPLICATION NOTE

All assays were conducted in Jurkat cell
medium. NK-92 cells were used in CMC
assays 48 hours after passage into new
medium. Signal transduction inhibitors
PP2, U73122, Sunitinib, and SP600125
were obtained from Sigma. Each inhibitor
was solubilized in 100% DMSO (various
concentrations) and stored at -20°C until
use.
CMC Titration Assays: The iQue Screener
CMC workflow is illustrated in Figure 1.
Prior to the start of the assay, Jurkat cells
(target) were encoded using the MultiCyt®
FL4 Cell Proliferation and Encoder Kit
(Intellicyt) according to the protocol.
Briefly, the cells were prepared in batch
and excess dye removed by washing
before use in the assay. The encoding
protocol takes approximately 20 minutes.
The following steps were performed in a
single 384-well plate with thirty replicates
of each condition and a total assay volume
of 20 μL/well. Unlabeled NK-92 cells were
serially diluted (1:2) over 12 steps from
60,000 cells/well down to 30 cells/well. At
the start of the assay, each dilution was
mixed with labeled Jurkat cells (6,000
cells/well) and incubated for four hours
at 37°C. The resulting effector:target
ratios ranged from 10:1 down to 1:200. To
assess CMC, we monitored two different
readouts- apoptosis using the Caspase
3/7 reagent from the MultiCyt Apoptosis
Kit (Intellicyt), and cell viability using the
MultiCyt FL3 Cell Membrane Integrity
reagent (Intellicyt). Both reagents were
15

| January, 2019

Figure 1. Three-step iQue Screener cell
mediated cytoxicity (CMC) assay workflow.


https://www.intellicyt.com/

Multiplexing Phenotype and Function for More Biologically Relevant Insights

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