Optimization of a flow cytometric method for the simultaneous measurement of cell surface antigen, DNA content, and in vitro BrdUrd incorporation into normal and malignant hematopoietic cells.
We have designed an assay for the simultaneous measurement of cell surface phenotype, S-phase fraction, and DNA content by single laser instrumentation for the purpose of determining the labeling index (LI), duration of S-phase (Ts), and the potential doubling time (Tpot) of leukocyte subpopulations. The procedure was optimized with regard to: mode of bromodeoxyuridine (BrdUrd) incorporation, selection of suitable leukocyte differentiation antigens (LDAs) as well as PE-conjugated monoclonal antibodies (MoAbs) against myeloid cells, overnight permeabilization and fixation (paraformaldehyde 1% and 0.05% Nonidet P40), DNase I treatment (250 Kunitz units), concentration of FITC-conjugated anti-BrdUrd MoAb (dilution 1:5), and DNA staining with 7-amino-actinomycin (7-AAD) (10 microg/ml). We validated this assay by measuring LI, Ts, and Tpot repeatedly in four leukemic cell lines and found these to be stable (coefficients of variation (CV): 0.06, 0.13, and 0.08, respectively). Finally, we employed the assay on different leukocyte preparations from normal donors (including purified CD34 + cells) and patients with malignant myeloid disorders, and we concluded that it will yield valuable data regarding the cell cycle kinetics of subsets of leukocytes in heterogeneous mixtures of hematopoietic cells.
Holm
M
M
Department of Medicine and Hematology, Aarhus University Hospital, Denmark.
Thomsen
M
M
Høyer
M
M
Hokland
P
P
D000208:Acute Disease; D000911:Antibodies, Monoclonal; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000737:chemistry*; D001973:Bromodeoxyuridine; D002463:Cell Membrane Permeability; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D003609:Dactinomycin / Q000031:analogs & derivatives; D005434:Flow Cytometry / Q000379:methods*; D006680:HLA Antigens / Q000032:analysis*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D009190:Myelodysplastic Syndromes / Q000097:blood* / Q000473:pathology; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D004396:Coloring Agents; D006680:HLA Antigens; D003609:Dactinomycin; D004247:DNA; D001973:Bromodeoxyuridine