Comparison of the supravital DNA dyes Hoechst 33342 and DAPI for flow cytometry and clonogenicity studies of human leukemic marrow cells.
Human leukemic bone marrow cells were studied by flow cytometry and a colony-forming assay. Two supravital DNA dyes, Hoechst 33342 (H33342) and 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI), were compared in terms of DNA histograms by flow cytometry and toxicity to cells by colony-forming assay. Initially, Chinese hamster ovary (CHO) cells were used, and the optimal staining conditions for the two dyes were determined: 30 min exposure to 10 micrograms/ml at 37 degrees C for H33342 and at 23 degrees C for DAPI. DAPI demonstrated DNA profiles with better coefficients of variation for Go/G1 cells than did H33342. This difference was consistently shown in four additional mammalian cell lines and bone marrows freshly obtained from five patients, four of which were leukemic. Both dyes, in the optimal staining conditions, can suppress the growth of CHO cells with H33342 more toxic than DAPI. In experiments on three leukemic bone marrows, H33342 was shown to be more toxic than DAPI in terms of colony-forming capability. Although there is considerable variation in the degree of the toxicity between different cases, more than 50% of leukemic colony-forming cells can survive after DAPI staining. These data indicate that DAPI is preferable to H33342 for use with human leukemic cells because the staining technique required is less stringent; there is a more homogenous staining of the DNA, and there is less cytotoxicity induced. Supravital staining of DNA with DAPI and viable sorting by flow cytometry should be reasonably possible for functional studies such as colony formation after sorting.
D000208:Acute Disease; D000818:Animals; D001562:Benzimidazoles / Q000633:toxicity; D001853:Bone Marrow / Q000473:pathology*; D001943:Breast Neoplasms; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000633:toxicity; D006801:Humans; D007211:Indoles / Q000633:toxicity; D007938:Leukemia / Q000473:pathology*; D010053:Ovary; D014410:Tumor Stem Cell Assay
D001562:Benzimidazoles; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride