A novel modification of a flow cytometric assay of phosphorylated STAT1 in whole blood lymphocytes for rapid detection of interferon-alpha signal in vivo.
Phosphorylation of signal transducer and activator of transcription factor 1 (STAT1) is a key response in the type I interferon (IFN) signal cascade. We developed a novel flow cytometric assay for phosphotyrosine-STAT1 (p-STAT1) to rapidly monitor in vivo IFN signaling. Mouse blood stimulated with mouse IFN-alpha was hemolyzed with lysis buffer in place of lymphocyte purification, permeabilized with methanol, and stained with an Alexa Fluor 488-conjugated anti-p-STAT1 antibody. The cells were also stained with phycoerythrin (PE)-conjugated anti-CD45 antibody for eliminating debris (CD45-negative) from leukocytes (CD45-positive), and with PE covalently linked to cyanin 5-conjugated anti-Gr-1 antibody for separating lymphocytes (Gr-1-negative) and granulocytes (Gr-1-positive). When whole blood was treated with IFN-alpha, the Alexa Fluor 488 intensity of lymphocytes increased, reaching a peak within 1 h, and this increase was statistically significant at IFN-alpha concentrations of 100 U/mL and higher. When IFN-alpha was administered intravenously to mice, the Alexa Fluor 488 intensity of blood lymphocytes was increased, reaching a peak in 1 h and returning to baseline at 18 h, and this increase was dose-dependent, with statistically significant increases seen at doses of 1,000 U/body and higher. The kinetics and dose-responses of p-STAT1 levels in the spleen, lung, and liver were similar to those in blood lymphocytes. This new flow cytometric assay of p-STAT1 in peripheral blood leukocytes will be useful for examining IFN-alpha signaling and for monitoring tissue response to IFN-alpha in vivo.
Tochizawa
Shiro
S
Research Institute of Pharmacological & Therapeutical Development, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0192, Japan. s_tochizawa@research.otsuka.co.jp
Ohmoto
Yasukazu
Y
Mori
Toyoki
T
D000818:Animals; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D006098:Granulocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D016898:Interferon-alpha / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D008214:Lymphocytes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D010766:Phosphorylation / Q000187:drug effects; D050794:STAT1 Transcription Factor / Q000032:analysis* / Q000097:blood; D013154:Spleen / Q000737:chemistry / Q000378:metabolism
D016898:Interferon-alpha; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse