M. Kubbies

BrdU—Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis☆

Unlike other techniques, flow cytometric analysis of BrdU-quenched 33258 Hoechst fluorescence may be used to measure cell activation and the G1, S, and G2/M compartment distributions in each of three successive cell cycles after growth stimulation of human peripheral blood lymphocytes. Cell cycle kinetic curves can be constructed from the BrdU-Hoechst flow data which allow the simultaneous assessment of growth fraction, lag-time, compartment exit rate, compartment duration, and compartment arrest. Applications of this new versatile technique include the evaluation of drug and growth factor effects, cell aging, and diagnosis in medicine and immunology.

Cell cycle kinetics by BrdU-Hoechst flow cytometry: an alternative to the differential metaphase labelling technique.

Abstract. Human lymphocytes cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. the BrdU‐Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non‐cycling cell fractions and transition probabilities. the Smith and Martin model, modified to include a fraction of non‐cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4‐6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non‐cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. the potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.

The phytohemagglutinin response of human peripheral blood lymphocytes as a function of donor age: A re-examination using BrdU-Hoechst flow cytometry

Mononuclear cells were isolated from peripheral blood by a standard Ficoll-Hypaque technique from 127 healthy donors, ranging in age from newborns to 86 years of age. As a measure of their in vitro growth response, the fraction of non-cycling cells was determined at 48 and 72 h after phytohemagglutinin (PHA) exposure by means of BrdU-Hoechst flow cytometry. This technique provides an optimal assay system for the non-cycling cell fraction, since all cycling cells will have incorporated BrdU thereby quenching the fluorescence of the Hoechst 33258 fluorochrome. Lymphocytes from prepubertal donors showed significantly decreased non-cycling cell fractions, as did lymphocytes from an additional group of 14 adults with hypogonadism due to the 45, XO condition (Turner-Syndrome). Much to our surprise, we found no definitive correlation between donor age and the non-cycling fraction of cells from the adult lymphocyte donors. Nor did we find any age-related increase in the variance of the non-cycling cell fraction. These observations suggest that the previously reported age-related decline in the PHA response of human PBL may reflect an increasing delay, rather than an overall diminution, of the PHA response as a function of donor age.

Interphase cell flow cytometry as a means of monitoring genomic size in normal and neoplastoid cell cultures

The DNA specific fluorescence of mass cultures and clones derived from human skin and bladder tumor tissue was assayed by flow cytometry. In order to detect and quantitate small fluorescence intensity changes, cytogenetically defined triploid or diploid human fibroblast strains were cocultivated, harvested, and stained with the cell strain of unknown karyotype. The triploid standard (derived from human abortus tissue) proved chromosomally unstable at high passage level. Fifteen male, female, and 45,X strains displayed target-to-standard cell fluorescence ratios commensurate with their respective chromosome constitutions. Interstrain variation was highest among the 45,X strains, although mosaicism could not be detected by conventional cytogenetics. Interclonal fluorescence variation was two- to ten-fold higher among the tumor-derived clones tested. Chromosome counts and subcloning experiments indicate that this increased fluorescence variation is due to genome size variation. The clonal evolution of genome size differences was observed in subclones of chromosomally divergent parental clones. These observations suggest that well controlled flow cytometry can adequately resolve subtle degrees of genome size variation in cultivated human cells. The technique is especially suited for monitoring genome size changes in cultivated tumor cells.

Analysis of Cell Cycle Distribution of Human Bladder Cancer Cells by BrdU-Hoechst Flowcytometry

The natural history and therapy of bladder tumors are dictated primarily by the stage and histopathology of the disease. Nevertheless, among this tumor type there is a broad spectrum of biological behavior, and its response to a particular treatment regimen is highly variable. One reason for this heterogeneous biological behavior could arise from differences in cell cycle kinetics. Traditionally, in vitro growth and cell cycle kinetics of bladder tumors have been investigated by mitotic chromosome labelling techniques, such as radioactive thymidine uptake (Morimoto et al. 1980) or BrdU-incorporation (Hefton et al. 1980). Except for an accurate but time-consuming double-labelling technique (Schultze 1981) these standard methods do not provide exact estimates of the growth fractions. Moreover, the identification of kinetically divergent subpopulations within the heterogenic bladder tumor cell culture has not been possible. The introduction of monoclonal BrdU antibodies have improved the estimates of S-phase cell fractions, but there is no evidence that this new technique can differentiate between phase fractions which belong to successive cell cycle generations. Here, we introduce an alternative technique which likewise uses the principle of BrdU-incorporation, but which detects BrdU-substitution by its quenching effect on Hoechst-dye fluorescence (Latt et al. 1977).