Evaluation of Stromal Cytokine-Induced Intracellular Protein Phosphorylation Using Phospho-Flow Cytometry

Take leukemia cells in growth media without stimulating factors such as cytokines, and incubate them.

The absence of stimulating factors allows intracellular phosphorylated proteins to undergo dephosphorylation and return to their native states.

Centrifuge and remove the media. Resuspend cells in stroma-supernatant containing the cytokine, thymic stromal lymphopoietin, or TSLP.

TSLP binds to its receptor on the leukemia cell, triggering receptor dimerization.

This brings the two Janus-associated kinases, or JAKs, closer, facilitating mutual transphosphorylation .

The activated JAKs phosphorylate the cytoplasmic tails of the receptor, creating docking sites for signal transducer and activator of transcription, STAT monomers.

Once phosphorylated, STATs dimerize and detach from the receptor to regulate downstream cellular processes.

Fix the cells to maintain the phosphorylation states of intracellular proteins.

Treat the cells with a permeabilization solution to increase the cell membrane permeability.

Add fluorescently labeled antibodies to label the phosphorylated proteins.

Flow-cytometric measurement of fluorescence signal correlates with TSLP-induced STAT phosphorylation.

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.