Detection of intracellular phosphorylated protein tyrosine kinases by flow cytometry

Objective: To set up the method of detection of intracellular phosphorylated protein tyrosine kinases(PTKs) by flow cytometry.Methods: After stimulated with PMA and PHA,Jurkat cells were fixed and permeabilized by two different methods,and the phospho-tyrosine monoclonal antibody,P-Tyr-100,and a FITC labeled second antibody were used to detect intracellular phosphorylated PTKs by flow cytometry.Correspondingly the Western-blotting method was also used to compare with flow cytometry.Two-colour immunofluorescence labelling technique was further used to detect phosphorylated PTKs in Mtb-Ag activated γδT cells of peripheral blood mononuclear cells(PBMC).Results: Phosphorylated PTKs in Jurkat cells increased obviously after stimulated with PMA and PHA.The results of flow cytometry were consistent with those of Western-blotting.By using two-colour immunofluorescence labelling technique,the phophorylated PTKs was detected preferentially in γδT cells among PBMCs stimulated with Mtb-Ag.Conclusions: The intracellular phosphorylated PTKs could be detected in single cell by flow cytometry.