BrdU-Hoechst-ethidium bromide (EB) quenching technique for studying kinetics of hematopoiesis.
Explore the source record for details and available documents.
Antigens, CD↗
Biomedical subjects
M Kubbies
Publications and source records attributed to M Kubbies.
Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain.
Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics.
Animals↗
Improved BrdUrd-Hoechst bivariate cell kinetic analysis by helium-cadmium single laser excitation.
In laser based flow cytometers, UV excitation of Hoechst 33258 and propidium iodide (PI) or ethidium bromide (EB) is performed with 351/364 nm high power lines of UV-capable argon ion lasers, which are expensive and short-lived. In this paper we note for the first time that helium-cadmium lasers emitting 10 to 30 mW at 325 nm are even more superior for cell kinetic bivariate bromodeoxyuridine (BrdUrd)/Hoechst PI or EB cell cycle analysis. HeCd single laser UV excitation gives comparable CVs for cell cycle distributions, and almost normal G2M/G1 ratios of 1.9 to 2.0 for all cell cycles. This is shown for synchronous and asynchronous cell populations on a FACStar+ and an Ortho Cytofluorograf. Therefore we recommend helium-cadmium lasers as low-power, cheap, and long-lived UV excitation sources for the cytochemically simple but high resolution multiparameter BrdUrd-Hoechst cell kinetic analysis.
Animals↗
Flow cytometric DNA-histogram analysis: non-stoichiometric fluorochrome binding and pseudoaneuploidy.
Detection of aneuploid subpopulations using flow cytometry requires stoichiometric binding of nucleic acid-specific fluorochromes onto DNA. It is shown that parameters like cell type specificity and differentiation stage, cell cycle stage, loss of DNA-integrity, cell preparation, and cytochemistry affect fluorochrome binding to DNA and give rise to the appearance of pseudo-aneuploid cell populations. Intercalating as well as non-intercalating fluorochromes show non-stoichiometric DNA-labelling in cell populations with identical DNA content, and pseudo-aneuploidy was found in flow cytometers equipped with either arc lamps or argon lasers. Pseudo-aneuploidy was never observed with intercalating and non-intercalating fluorochromes within identical specimens, consisting of cells of various differentiation states (e.g., bone marrow) or containing large numbers of dead cells. Therefore, fluorochromes exhibiting different base-pair specificities or steric binding modes should be applied to be sure of the correct interpretation of small levels of hypo- or hyper-diploidy (+/- 20 per cent).
Amniotic Fluid↗
Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.
We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).
Animals↗
Alteration of cell cycle kinetics by reducing agents in human peripheral blood lymphocytes from adult and senescent donors.
For improving cell proliferation reducing agents are routinely used as medium supplements in murine cell cultures, however, they are rarely used for human peripheral blood lymphocytes (PBLs). Data on changes in cell kinetics induced by reducing agents are not available. Here cell kinetic alterations induced by reducing agents in human lymphocytes are revealed by applying flow cytometric BrdUrd/Hoechst cell cycle analysis and by using the exit kinetic model of Smith and Martin. Applying alpha-thioglycerol (a-TG) as a model compound it was shown that the major cell kinetic effect is a shortening of the mean duration of the G0/G1 phase. The minimum G0/G1 phase duration and the percentage of the non-cycling G0/G1 cell fraction decrease only slightly. Moreover, a lower number of PBL's are arrested in the G2/M phase of the 1st cell cycle. The durations of the S and G2/M phase in the 1st and G1 phase in the 2nd cycle are not affected. These cell kinetic effects are identical for lymphocytes from both adult and senescent donors. The supplementation of the cell cultures with recombinant IL-2 did not induce similar cell kinetic alterations compared with a-TG. This indicates that the variation of the cell cycle progression factor IL-2 is not solely responsible for improvement of the cell activation process in the G0/G1 phase.
Aging↗
UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.
Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle distribution. With the HeCd laser, high-resolution cell kinetic analysis applying the novel BrdU/Hoechst-PI quenching technique showed superior resolution and an almost normalized G2M/G1 channel ratio of the first cell cycle. Indo-1 analysis for detection of intracellular free calcium gave similar results for both excitation sources, although the indo-1 ratio of activated cells was lower for HeCd excitation. Monochlorobimane as an indicator fluorochrome of glutathione content could not be excited sufficiently with the 325-nm line of the HeCd laser and exhibited poor resolution between positive and negative cells. However, the second glutathione-specific fluorochrome o-phtalaldehyde gave even better results with the HeCd laser. Our data indicate that air-cooled HeCd lasers are cheap and reliable UV-excitation sources for most UV-excitable fluorochromes, and might be an alternative to the expensive water-cooled argon and krypton laser.
Argon↗
Rat "first-wave" mature thymocytes: cycling lymphoblasts that are sensitive to activation-induced cell death but rescued by interleukin 2.
The sequential appearance of thymocyte subsets in rat ontogeny was studied using the surface markers CD4, CD8, and the alpha/beta T cell receptor (here referred to as TcR). It was noted that the first TcRhigh thymocytes, appearing around birth, are not resting lymphocytes but cycling blast cells. These "first wave" TcRhigh cells are medullary in location and predominantly of the CD4/CD8 "single-positive" phenotype. Only about 5% express the light chain of the interleukin (IL)2 receptor, indicating that binding of IL 2 to high-affinity receptors is not driving proliferation of these blasts. Newborn TcR high blast cells were purified and analyzed in vitro. When cultured without further additions, they rapidly stopped cycling. Stimulation with cross-linked anti-TcR monoclonal antibody plus IL 2 resulted in vigorous and rapid proliferation that exhibited accelerated kinetics as compared to peripheral resting T cells. In contrast, TcR cross-linking without exogenous IL 2 induced cell death. This TcR-induced cell death involved fragmentation of DNA characteristic of apoptosis that was readily detectable within 18 h of culture. Addition of IL 2 to these short-term cultures prevented TcR-induced DNA fragmentation. Together, these results suggest that in newly formed TcRhigh thymocytes, TcR engagement results in clonal deletion if the IL 2 receptor remains unoccupied but allows clonal expansion if IL 2 is provided. This mechanism may be operative in the establishment of self-tolerance during T cell development.
Animals↗
Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes.
Cis-platinum (CDDP) induces severe inhibition of cell activation and cell cycle progression in PHA-stimulated human PBL's. Applying the novel BrdU/Hoechst flow cytometric technique for high resolution cell cycle analysis we show that CDDP induced multiple cell kinetic disturbances occur simultaneously comprising G0/G1-arrest, and slow down and arrest of cells in S and G2/M-phase. We investigated whether the administration of reduced glutathione (GSH) might rescue cells from proliferative disturbances induced by CDDP. GSH at 0.15 mg ml-1 only partially restored normal cell activation and cell cycle progression. However, at 1.5 mg ml-1 a complete normal proliferation pattern was obtained. At the highest GSH dose rescue from inhibition of cell activation (G0/G1-phase arrest) as well as of cell cycle progression (S- and G2/M-phase arrest) was also present after delayed addition of GSH (1, 4 and 20 h) to CDDP treated PBL's. In addition cell viability of CDDP exposed PBL's is restored after GSH treatment. Our in vitro experiments give evidence that an increase of WBC found in CDDP/GSH treated patients has a real underlying cellular physiological mechanism protecting human peripheral lymphocytes from CDDP toxicity.
Bromodeoxyuridine↗
Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding.
Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X-rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non-cycling cell fraction; and a distinctive, non-cycling G-/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst 33258-ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non-cycling cell fraction in the control culture to 29% (X-rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3-aminobenzamide, this aberrant cell population increased significantly after X-ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non-cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non-cycling and cycling cell fractions, this subpopulation with non-stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of non-cycling cells represents damaged cells with altered dye binding properties.
Adult↗
Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry.
The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 microM methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during G1 and/or at G1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G2/M. To determine t-PA production CHO cells were sorted from G1-, early/late S-, and G2/M-phase. After 1-, 2-, and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production and secretion (2-h assay), unlike the expression of DHFR, increased continuously from relatively 100% in G1 to 127% in early S and reached its maximum of 159% in late S, whereas in G2/M-phase it decreased to 118%. Our results show that in pSVtPA.dhfr-coamplified CHO cells gene products DHFR and t-PA both exhibit different cell cycle-correlated accumulation and secretion, respectively, indicating that the brightest MTX-FITC-positive cells (G2/M) do not display the highest t-PA secretion rate.
Animals↗
Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells.
Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.
Animals↗
Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258-ethidium bromide bivariate flow cytometry.
Explore the source record for details and available documents.
Animals↗
Improvement of human lymphocyte proliferation and alteration of IL-2 secretion kinetics by alpha-thioglycerol.
Using a novel flow cytometric assay (BrdU/Hoechst flow cytometry) for the visualization and quantitation of cell activation and cell progression through multiple cell cycles we show that (1) resting mononuclear cells from human venous blood respond to polyclonal activation in a highly asynchronous fashion, as some of these cells enter their first cell cycle as early as 30 and as late as 80 hrs after exposure to the mitogen; (2) reducing agents improve the mitogenic response of polyclonally activated human lymphocytes by increasing the recruitment of non-cycling G0/G1 cells; (3) of 8 reducing agents tested, alpha-thioglycerol proved most effective with respect to enhancing recruitment into the cell cycle; (4) the effective agents needs to be present permanently for maximum response; (5) the growth-promoting effect of alpha-thioglycerol is mediated, but not solely caused by increased IL2 production. The latter conclusion was arrived at by using the IL2-dependent CTLL cell line in conjunction with the BrdU/Hoechst flow technique as a sensitive bioassay system for the growth promoting activity of the cell cycle progression factor IL2; (6) exogenously added IL-2, however, did not improve cell activation towards proliferation. These data indicate that increased IL-2 levels are probably due to increased cell activation rates but are not the primary cause of improvement of human lymphocyte proliferation.
Adult↗
Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/Hoechst flow cytometry in cell kinetics.
Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5-bromodeoxyuridine (BrdUrd) offers such a potential. We illustrate here that the bivariate analysis of non-BrdUrd-quenched ethidium bromide vs. BrdUrd-quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.
Bisbenzimidazole↗
Complex Ca2+ flux inhibition as primary mechanism of staurosporine-induced impairment of T cell activation.
The inhibitory effect of the highly effective drug staurosporine on the early activation signal Ca2+ flux was investigated via multiparameter flow cytometry in human peripheral blood T lymphocytes. Staurosporine has been reported to be a specific inhibitor of protein kinase C. However, we show that it inhibits the Ca2+ influx in anti-CD3 and phytohemagglutinin-stimulated human CD4+ and CD8+ lymphocytes at concentrations between 1.0 and 10.0 ng/ml. Staurosporine decreases the number of Ca2+-positive CD4+ and CD8+ lymphocytes as well as the Ca2+ influx per cell; the drug also delays the time of the maximum response to polyclonal stimulation. In addition, we demonstrate that staurosporine affects the primary Ca2+ response via inhibition of the release of the membrane-bound Ca2+ from the endoplasmic reticulum in CD4+ and CD8+ lymphocytes. Binding studies of the anti-CD3 antibody to T lymphocytes indicate normal binding capacities in the presence of staurosporine. With respect to the classical scheme of T cell activation via phospholipase C, our data suggest that staurosporine may inhibit T cell activation primarily by its effect on the early Ca2+ flux signal.
Alkaloids↗
Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation.
The BrdUrd-Hoechst method was used to analyze the interaction of various oxygen concentrations with BrdUrd substituted DNA with respect to cellular proliferation. At oxygen concentrations above 5%, human diploid fibroblast-like cells and amniotic fluid fibroblast-like cells showed reduced proliferation rates, which resulted from an increase in noncycling cells and from a permanent arrest of cells in the G2 phase of the cell cycle. At 35% oxygen the increase in noncyling cell fraction and the permanent arrest in G2 was strongly dependent upon the concentration of BrdUrd. Incorporation of BrdUrd into DNA, therefore, amplifies the adverse effects of increasing oxygen concentrations upon cell proliferation. The mechanism of this amplification might involve a free radical attack on DNA similar to the radiation sensitizing effect of BrdUrd.
Bromodeoxyuridine↗
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.
Benzimidazoles↗