DISCRIMINATION OF BROMODEOXYURIDINE LABELED AND UNLABELED MITOTIC CELLS IN FLOW CYTOMETRIC BROMODEOXYURIDINE DNA ANALYSIS/

Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, resp ectively, can be discriminated from interphase cells using a new metho d, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forw ard and orthogonal light scatter. The method was optimized using the h uman leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fix ed cells were treated with pepsin/HCl and stained as a nuclear suspens ion with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could be discriminated from unlabe lled mitoses, and from labelled and unlabelled G(2) cells, by their in termediate log FITC fluorescence intensity. In addition, mitoses and G (2) nuclei differed in forward and orthogonal light scattering, but ha d equal intensity of propidium iodide fluorescence. This method for di scrimination of labelled mitoses was also tested on cultured normal ad ult human keratinocytes labelled with iododeoxyuridine (IdUrd). In ker atinocytes, where the cell structure was preserved after pepsin/HCl, I dUrd labelled mitotic cells were similarly discriminated in the log FI TC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses. (C) 1994 Wiley-Liss, Inc.