To validate the supernatant produced from engineered stroma, thaw the supernatant samples harvested from the control in cytokine-expressing stroma, and plate 3 wells of leukemia cell lines per condition in R20 medium at a 2 times 10 to the fifth cells per well concentration in a 96-well tissue culture plate. Incubate the cells for two hours at 37 degrees Celsius to allow for the phosphorylation from their prior culture to be lost.
Then, transfer the cells from each well into individual 4-milliliter tubes, and collect the cells by centrifugation. Resuspend the pellets in 600 microliters per experimental condition, and plate 200 microliters of cells per well for each condition in triplicate. Incubate the cells for the appropriate period according to the specific commercial phospho-assay.
At the end of the incubation, pellet the cells by centrifugation, and label the cells via phospho-flow cytometry staining according to the manufacturer's protocol. Then, analyze the cells by flow cytometry using standard flow cytometric analysis protocols to verify that cytokine in the stroma supernatant phosphorylates the leukemia cells.
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