Multiplexing Phenotype and Function for More Biologically Relevant Insights - 18

More Biologically Relevant Insights | APPLICATION NOTE

that are detected as FL3-positive, and therefore reports on the number of nonviable cells, whereas the lower-intensity "negative" peak is comprised of the viable
cells that have excluded the dye and are not fluorescent (Figure 3C, left peak).
Finally, the same analysis can be done on the NK-92 cells to verify that they remain
unaffected by incubation with the target cells (Figure 3D, E). In this case, the same
gates used to assess activation of caspase 3/7 (FL1) and cell viability (FL3) for Jurkat
cells were applied to the NK-92 cells for an unbiased analysis.
Note that for this example we chose to detect caspase 3/7 activation and cell
membrane integrity, but a number of other reporters can be simultaneously
detected and quantified for each cell type. Examples of other MultiCyt reagents
that have been validated in multiplex with either the caspase or the cell viability
reagents include Annexin V binding, mitochondrial membrane depolarization
(MultiCyt Apoptosis Kits), cell proliferation (MultiCyt Cell Proliferation and Encoding
Kit), and detection of secreted cytokines (QBeads® PlexScreen and DevScreen Kits).
Reproducible quantification demonstrated by effector cell titration: To demonstrate the ability of the iQue Screener CMC assay to provide highly quantitative
information, we assessed the dose-response relationship of NK-92 effector cells on
CMC of Jurkat target cells. Keeping the number of Jurkat cells fixed at 6,000 cells/
well, we added increasing amounts of NK-92 cells, changing the effector:target
ratio from 1:200 to 10:1(Figure 4). Each data point is the average of measurements
from 30 different wells, with error bars as indicated.
Examination of the caspase 3/7 activation and cell viability plots for the NK-92
cells show background levels of caspase activation and cell viability, with higher
variability at the lower NK-92 concentrations likely a reflection of measurement
uncertainty at extremely low cell densities.
Assay sensitivity, cell type discrimination demonstrated by CMC inhibitor doseresponse assay: To demonstrate the sensitivity and specificity of the iQue Screener
CMC assay, we performed the assay in the presence of increasing amounts of
known CMC inhibitors that target different signaling pathways (Figure 5)-sunitinib (tyronsine kinase inhibitor), PP2 (Src inihibitor), U73122 (PLC inhibitor), or
SP600125 (JNK inhibitor). Note that the ability to observe the effects of pathwayspecific inhibitors on both effector cells and target cells helped us to quickly
18

| January, 2019

Figure 4. CMC Titration Assay. Cell viability (A) and Caspase 3/7 activation (B) assessment
demonstrate that Jurkat target cells are killed by NK92 effector cells in a dose-dependent
manner. For each data point, n = 30 with the curves simply connecting each data point.
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
