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High‐throughput evaluation of Janus Kinase‐STAT and MAPK pathways in normal peripheral whole blood cells using 96 deep well plates by flow cytometry
Author(s) -
Sisouvanthong Chad,
Ernst David,
Li Li,
Wang Xiao,
Chen Jie,
Shih Jing Ping,
Huang Joseph,
Gao Guojian
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1064.17
Subject(s) - flow cytometry , peripheral blood mononuclear cell , microbiology and biotechnology , whole blood , lysis buffer , intracellular , kinase , cell , biology , janus kinase , phosphorylation , lysis , chemistry , immunology , biochemistry , in vitro
When performing high‐throughput, flow cytometric screening of the patterns by which treated cells induce phosphorylated proteins involved in signaling pathways, tedious preparation of peripheral blood mononuclear cells (PBMC) was usually required. However, we have developed a Whole Blood Lyse/Fix Buffer System. This system allows for simultaneous red blood cell lysis and the fixation of leukocytes human whole blood. The cells can be stained for cell surface markers and intracellular cell signaling molecules and subsequently analyzed by flow cytometry. This eliminated the need for preparing PBMC. We have recently extended the application of this Whole Blood Lyse/Fix Buffer System to process cell samples in 96‐deep‐well plates. This method greatly facilitates the ease of preparing multiple human whole blood samples for cell signaling flow analysis. Whole blood stimulation, fixation, permeabilization, staining, and acquisition were done in deep well plates. Using deep well plates, we were able to evaluate the dose responses and time courses of STAT1, STAT3, STAT5, STAT6, Erk1/2, and P38 phosphorylation expressed by human whole blood cells that received various treatments. Inhibitory reagents, such as MEK and JAK inhibitors, were also applied to look at their differential effects on the phosphorylation status of intracellular signaling molecules that can be modified by the targeted kinases.