A novel modification of a flow cytometric assay of phosphorylated STAT1 in whole blood lymphocytes for rapid detection of interferon-α signal in vivo

摘要: Abstract Phosphorylation of signal transducer and activator transcription factor 1 (STAT1) is a key response in the type I interferon (IFN) cascade. We developed novel flow cytometric assay for phosphotyrosine-STAT1 (p-STAT1) to rapidly monitor vivo IFN signaling. Mouse blood stimulated with mouse IFN-α was hemolyzed lysis buffer place lymphocyte purification, permeabilized methanol, stained an Alexa Fluor 488-conjugated anti-p-STAT1 antibody. The cells were also phycoerythrin (PE)-conjugated anti-CD45 antibody eliminating debris (CD45-negative) from leukocytes (CD45-positive), PE covalently linked cyanin 5-conjugated anti-Gr-1 separating lymphocytes (Gr-1-negative) granulocytes (Gr-1-positive). When whole treated IFN-α, 488 intensity increased, reaching peak within 1 h, this increase statistically significant at concentrations 100 U/mL higher. administered intravenously mice, 1 h returning baseline 18 h, dose-dependent, increases seen doses 1000 U/body kinetics dose–responses p-STAT1 levels spleen, lung, liver similar those lymphocytes. This new peripheral will be useful examining signaling monitoring tissue vivo.