Multiplexing Phenotype and Function for More Biologically Relevant Insights - 16

More Biologically Relevant Insights | APPLICATION NOTE

used as stated in the product protocols, and were added to the reaction at the same
time as the NK-92 cells. After four hours, the 384-well plate was placed directly into
the iQue Screener and analyzed.
CMC Inhibition Assays: The CMC inhibition assay was performed as described for
the CMC titration assay with the following modifications. A 16-step, 1:2 dilution
series of each inhibitor was prepared with the following as the highest concentrations: Sunitinib at 25 ng/ml; PP2 at 100 mM; U73122 at 37.5 mM; SP600125 at 150
mM. NK-92 cells (12,000 cells/well) were preincubated at 37°C for 20 minutes with
each dilution of inhibitor, and then mixed with Jurkat cells (6,000 cells/well). An
effector:target ratio of 2:1 was used for this study, with a total reaction volume of 20
μL. Each condition was performed in triplicate.
The Intellicyt® iQue Screener and ForeCyt® software: The Intellicyt® iQue Screener
PLUS provides high-throughput measurements using a flow cytometry detection
engine and patented air-gap delimited sampling technology, which can sample wells
with zero dead volume. Samples are delivered in a continuous stream to the detector
engine without needing to pause between each sample. Individual wells are separated by discrete air gaps ensuring accurate discrimination of each sample (Figure 2).
ForeCyt Software was used to acquire data, set appropriate gates, quantify, and plot
CMC titration and inhibition data as described in the Results and Discussion section.
Results and Discussion
CMC assay overview: The Intellicyt® iQue Screener PLUS platform CMC assay is a
simple, three-step process that enables rapid screening of multiple conditions (Figure
1). First, target cells are labeled with an encoding dye, and then incubated with
effector cells and reporter dyes, all in the same well of a multi-well plate. At the end of
the incubation, the plate is placed into the Intellicyt® iQue Screener PLUS for analysis.
The resulting data is analyzed using ForeCyt Software to set appropriate gates,
allowing discrimination of target cells from effector cells and quantification of cell
membrane integrity and caspase 3/7 activation in individual cell types.
The advantages of this assay stem not only from its easy implementation as a highthroughput screen but also from the streamlined protocol, which reduces hands-on
time. By adding staining reagents at the beginning of the assay, the labor involved in
downstream steps is minimized while still providing robust results.

| January, 2019

Figure 2. iQue® Screener Technology provides multiparameter, high-content analysis
with as little as 1 μL of sample and zero dead volume.

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Multiplexing Phenotype and Function for More Biologically Relevant Insights

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