Detection of megakaryocyte colonies in plasma clot cultures by immunoenzymatic staining.
In vitro induced megakaryocytic differentiation/maturation of megakayocyte (meg) progenitors represents an important tool for investigating cytokine-induced in vitro thrombocytopoiesis. We have developed an assay which allows the in situ study of human meg progenitor-derived colonies, cultured on a plasma clot in the presence of cytokines. Plates were immunostained by using an anti-alpha IIb beta 3 monoclonal antibody and an alkaline phosphatase-labeled secondary antibody. alpha IIb beta 3-bearing cells were stained an intense red and were clearly differentiated from the negative cells. Processed plates were stable for some weeks at 4 degrees C. The described procedure is easy to perform and allowed us to enumerate the meg colonies and assess colony morphology and cell ploidy.
Perutelli
P
P
Pasino
M
M
Marotta
F
F
Mori
P G
PG
D003114:Colony-Forming Units Assay / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques; D008533:Megakaryocytes / Q000166:cytology*