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Preparation of reagents before assay
For the stimulation of blood samples prepare standard tissue culture test plate and add tested blood into individual wells together with appropriate reagents for negative control sample, positive control sample and for the sample to be stimulated with stimulus of your choice.
Visualize compensated data of negative control sample, positive control sample and stimulated sample in the sidescatter (SSC) versus CD45 FITC dot-plot. Set the gate around lymphocyte population. Visualize CD45+ lymphocytes in a dot-plot CD16+56 PE versus CD3 PE-Cy™5. Separate populations using appropriate gate of natural killer (NK) lymphocytes situated in upper-left quadrant (CD16+56+, CD3-) and T lymphocytes situated in right quadrants (CD3+) on the dot-plot. Finally, plot population of NK cells (CD16+56+, CD3-) as cell count (number of events) versus CD69 PE-Cy™7 in histogram. The data in the histogram for negative control sample are used to generate the discrimination gates defined as negative and positive in the first histogram. It is essential in order to set appropriate gates and calculate the percentage of CD69 positive NK cells (activated cells) in positive control sample and stimulated sample.