Evaluation of different fixation-permeabilization methods for simultaneous detection of surface, cytoplasmic markers and DNA analysis by flow cytometry in some human hematopoietic cell lines.
Different fixation/permeabilization methods were investigated for their convenience for simultaneous detection of membrane marker/DNA staining or cytoplasmic marker/DNA by flow cytometry. Nine different methods were employed. The expression of membrane marker CD20 and cytoplasmic marker CD22 on BJAB and DAUDI cells and cytoplasmic CD3 on MOLT4 cells were measured. Optimal methods were those that combine paraformaldehyde and saponin (for membrane CD20/DNA staining and cytoplasmic CD3/DNA staining) or buffered formaldehyde-acetone (for membrane CD20/DNA staining and for cytoplasmic CD22/DNA staining). Special interest was focused on proliferation marker CD71 and nuclear antigen Ki67. We investigated CD71 in MOLT4 cells and Ki67 in MOLT4, DAUDI and BJAB cells. Both of these markers are closely associated with proliferation rate. The optimal method for detection of Ki67/DNA staining combines paraformaldehyde with Tween 20.
Glasová
M
M
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia.
Koníková
E
E
Kusenda
J
J
Babusíková
O
O
D015703:Antigens, CD / Q000032:analysis*; D018951:Antigens, CD20 / Q000032:analysis; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D015815:Cell Adhesion Molecules; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D037102:Lectins; D007938:Leukemia / Q000235:genetics / Q000276:immunology*; D008223:Lymphoma / Q000235:genetics / Q000276:immunology*; D011990:Receptors, Transferrin; D051918:Sialic Acid Binding Ig-like Lectin 2; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; D017252:CD3 Complex; C068126:CD71 antigen; D015815:Cell Adhesion Molecules; D004273:DNA, Neoplasm; D037102:Lectins; D011990:Receptors, Transferrin; D051918:Sialic Acid Binding Ig-like Lectin 2