Preparation of positive control platelets for detection of canine platelet surface-associated IgG, IgM and complement (C3) by flow cytometry.
An assay for detection of platelet surface-associated (PSA-) IgG, IgM and/or complement (C3) in dogs was modified by preparation of artificial positive control platelets. Flow cytometry of fluorescein isothiocyanate (FITC)-conjugated anti-dog IgG, anti-dog IgM and anti-dog C3 antibodies was used to detect the PSA proteins. IgM single, IgM/C3 double and IgG/IgM/C3 triple positive platelets were prepared. FITC-conjugated anti-IgG antibody bound strongly only to the triple positive platelets. Binding of FITC-conjugated anti-IgM or anti-C3 antibody to the double and triple positive platelets was specifically blocked by preincubation with the respective non-FITC-conjugated same-origin antibodies. These results confirm that FITC-conjugated antibodies specifically detect PSA proteins and that the control platelets prepared in this study are appropriate positive controls for detection of PSA proteins by flow cytometry.
Tsuchiya
Ryo
R
Laboratory of Internal Medicine II, School of Veterinary Medicine, Azabu University, Kanagawa, Japan. tsutiyar@azabu-u.ac.jp
Komatsu
Takahiro
T
Ishikawa
Takefumi
T
Neo
Sakurako
S
McConnell
Mary F
MF
Hisasue
Masaharu
M
Yamada
Takatsugu
T
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology; D003176:Complement C3 / Q000378:metabolism; D004285:Dogs; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D007074:Immunoglobulin G / Q000097:blood*; D007075:Immunoglobulin M / Q000097:blood*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D010976:Platelet Count / Q000662:veterinary
D003176:Complement C3; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008070:Lipopolysaccharides; D016650:Fluorescein-5-isothiocyanate