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.