Identification of cell types in Sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

Identification of cell types in sertoli cell-enriched cultures by immunocytochemistry and DNA-specific fluorochrome Hoechst 33342

Summary The cell types in Sertoli cell-enriched cultures can be identified by using the DNA-specific fluorochrome Hoechst 33342 staining. This simple, rapid and reproducible procedure can be used with fixed and living cells. The peritubular myoid cells can be distinguished from the Sertoli cells in Sertoli cell-enriched cultures by the characteristic staining pattern obtained using Hoechst 33342 dye. Those cells identified as peritubular myoid cells by the characteristic DNA staining also interacted with the anti-fibronectin antibody determined by an immunocytochemical method while the Sertoli cells did not. The described staining method is valuable in assessing the presence of peritubular myoid cells in Sertoli cell-enriched cultures.     

ABCG2 is expressed in late spermatogenesis and is associated with the acrosome

An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72 kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.     

Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation

Accumulative evidence demonstrates that normal as well as cancer stem cells can be identified as a side population following Hoechst 33342 staining and flow cytometric analysis. This popular method is based on the ability of stem cells to efflux this fluorescent vital dye. We demonstrate that Hoechst 33342 can affect cell differentiation, suggesting potential complications in the interpretation of data.     

"DNA clearing" from non-covalently bound agents in mammalian cells as a new mechanism of drug resistance.

Earlier, we have described the process of active dissociation or "DNA clearing" from non-covalently bound agents in living mammalian cells. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds DNA in the minor groove tightly but non-covalently, was used for studying the interaction of non-covalently binding agents with DNA. Multiple drug resistance (MDR) in tumour cells is related to the expression of transport proteins that alter the cellular drug transport and distribution. Three different groups of genes (mdr, MRP, and LRP) and their products are implicated in MDR (A. Krishan, C. M. Fitz, and I. Andritsch, Cytometry 29:279-285 (1997)). To obtain new cell lines characterized by enhanced process of active dissociation of non-covalently bound agents from DNA or "DNA clearing", we carried out step-by-step selection with increasing concentrations of Hoechst 33342. The rodent cell lines hyperresistant to Hoechst 33342 and selected from AA8 were named AA8Hoe-R-1-AA8Hoe-R-10, and the cell lines selected from L cells were called LHoe-R-1-LHoe-R-10. The most resistant of them, AA8Hoe-R-6 and AA8Hoe-R-7, were able to grow in the presence of 80 microm/ml of Hoechst 33342 in the cell culture medium. All mutants were analyzed with the flow cytometric technique and were divided into two different groups. We conclude that the drug resistance of the first group of cell lines was due to changes in transport proteins. The second group of the resistant cell lines was characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. As we believe, the enhanced level of "DNA clearing" was caused by the amplification of some genes, because the gradual increase of Hoechst resistance in the same cell line resulted from the increase in the ability to remove the dye from DNA. These lines were shown to be also resistant to netropsin.     

Modification of the radiation sensitivity of human tumour cells by a bis-benzimidazole derivative

A comparison was made of the ability of either X-radiation or a DNA-specific ligand (the vital bis-benzimidazole dye; Hoechst 33342) to induce: cell killing, inhibition of de novo DNA synthesis, DNA strand breakage and the delay of cell division in human colon adenocarcinoma cells in vitro. Unlike radiation-induced cell killing, ligand-induced cytotoxicity appeared to be positively correlated with the extent of inhibition of de novo DNA synthesis--a feature consistent with the persistent binding of ligand molecules to nuclear DNA. Ligand-induced DNA strand-breaks disappeared slowly although ligand-treated cells retained apparently normal capacities to repair discrete radiogenic DNA strand-breaks. Pre-treatment of cells with Hoechst 33342 resulted in a dose-modifying enhancement of radiation resistance not associated with altered dosimetry for strand-break induction. However, radioresistance was accompanied by the protracted retention of cells in the G2 phase of the cell cycle. We suggest that the results provide direct evidence that the retention of cells in G2 phase is a sparing phenomenon and is triggered by the responses of chromatin domains to the presence of DNA damage. Our results have implications for the use of DNA-interactive agents in combined modalities for tumour therapy, and indicate a possible basis for the sparing of some tumour cells in dividing populations.     

Comparative accumulation of the Hoechst 33258 fluorescent probe in leukemia P388 cells sensitive and resistant to doxorubicin

The authors studied accumulation of the fluorescent probe Hoechst 33258 in leukemia P 388 sensitive (P 388/0) and resistant to doxorubicin (P 388/DOX) cells. It was shown that intensity of fluorescence of the dye increased after binding with nuclear DNA during 25 min for both lines of the cells. Intensity of fluorescence was 40% greater in sensitive than resistant cells. If Triton X-100 was added no difference between two lines of the cell was observed. When doxorubicin was added to the cells with dye, the intensity of fluorescence decreased. It was suggested to use Hoechst 33258 for assessment extent doxorubicin accumulation in nuclei of the cells.     

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Abstract

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.     

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.     

Molecular and functional properties of lung SP cells

Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under profibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.     

Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.     

DNA damage, cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro

This study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell-cycle phase distribution of L1210 cells. HO 33342 exposure for 2 h, at 37 degrees C produced DNA single-strand breaks as assessed by the method of alkaline elution. DNA single-strand breaks were concentration dependent (in the range .5-5 micrograms/ml) and increased significantly when HO 33342 (0.5-1.5 micrograms/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination. HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 microgram/ml--a concentration ten-fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the G2-M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.     

A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA stains

A comparison of four DNA stains considered to be AT-specific with chromosomes from a clonal Chinese hamster cell line B14F28-C5 have been made. The flow karyotype histograms indicate that DAPI, DIPI, and Hoechst 33258 and 33342 do stain similarly in the same preparation. DAPI staining is specific and highly reproducible in this line. We, therefore, recommend this dye as a single chromosome DNA stain for high-resolution flow cytometric measurements in cytogenetics and mutation research.     

A Comparative Study of Dapi, Dipi, and Hoechst 33258 and 33342 as Chromosomal Dna Stains

A comparison of four DNA stains considered to be AT-specific with chromosomes from a clonal Chinese hamster cell line B14F28-C5 have been made. The flow karyotype histograms indicate that DAPI, DIPI, and Hoechst 33258 and 33342 do stain similarly in the same preparation. DAPI staining is specific and highly reproducible in this line. We, therefore, recommend this dye as a single chromosome DNA stain for high-resolution flow cytometric measurements in cytogenetics and mutation research.     

A rapid method for flow cytometric cell counting and cell cycle analysis from monolayer cultures of tumor cells.

A rapid, simple, and reliable flow cytometric method using the histochemical fluorescent stain Hoechst 33342 in presence of the non-ionic detergent Triton X-100 has been reported. The processing of melanoma cell cultures to get nuclei stained with the fluorescent dye was accomplished in one step and within an hour permitted concurrent flow cytometric measurement of cell density and cell cycle analysis. The preparation is stable for more than three weeks at room temperature for flow cytometry. The histograms are reproducible and exhibit a coefficient of variation of less than 2.5% (G1 peak). The cell density measurements varied within +/- 5% limits.     

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.     

Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis and B. cereus: a flow cytometric study

For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA.     

DNA double staining for a fluorescence energy transfer study of chromatin in liver cells

Methodological aspects related to the application of techniques based on fluorescence energy transfer in the study of chromatin structure, were first examined. Fluorochromes specific for DNA with different interaction mechanisms were employed, both in single and double stainings. The following dye pairs were considered as donor/acceptor couples: Hoechst 33342 or DAPI/Mithramycin A or Chromomycin A3, Hoechst 33342 or DAPI/Propidium Iodide, and Mithramycin A or Chromomycin A3/Propidium Iodide. Spectrofluorometric analysis showed that the spectral distribution of the dye pair Ho/PI is more suitable for the evaluation of energy transfer efficiency. This dye pair was used in the study of the chromatin microstructure in rat hepatocytes isolated from livers at two different growth stages. In particular, diploid mono- and binucleated cells from young and adult rats were considered. The results indicated the existence of a more homogeneous situation in young than in adult rats. In the latter case, the statistical analysis indicates the presence of two groups of energy transfer values. The different efficiency values in energy transfer can be considered a consequence of chromatin structure rearrangement and are tentatively interpreted according to the functional role of the diploid cells in the two stages of liver growth.     

Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm

Hoechst 33342 is the fluorophore used routinely to measure DNA in X- and Y-chromosome-bearing mammalian sperm so they can be separated by flow sorting. A difference of <3% in DNA mass can be detected. This synthetic dye consists of two adjacent benzimidazole rings with N-methyl-piperazine and phenolic groups at the ends. The molecule permeates the cell membrane of living cells and binds selectively to A-T base pairs exposed in the minor-groove of double stranded DNA. Capability to distinguish and separate X- and Y-chromosome-bearing sperm has led to artificial insemination of somewhere around a million female mammals. Offspring with obvious abnormalities are no more frequent than after insemination of unsorted sperm into cows, horses, humans, pigs, sheep, rabbits, dolphins and other mammals. There is no apparent genotoxic effect from exposure of sperm to Hoechst 33342, although information on cellular toxicity or development of embryos resulting from Hoechst 33342-stained sperm is less reassuring. Little is known about the fate of sperm-delivered Hoechst dye in the female reproductive tract or on progeny of resultant offspring.     

Effects of Hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells

Our objective in this investigation was to determine whether Hoechst 33342, which is widely recognized as a DNA specific fluorochrome for living cells, is in fact nontoxic and kinetically nonperturbing at dye concentrations required to achieve acceptable DNA distributions. Three cell types were tested: HeLa S-3; SK-DHL2, a human lymphoma cell line; and hematopoietically normal human bone marrow cells. In the third system, only the cloning efficiencies were determined. Results differed considerably for the different cell types. While HeLa cells yielded excellent DNA distributions and were almost completely resistant to the cytotoxic and cytokinetic effects of the dye, SK-DHL2 cells were highly sensitive to the fluorochrome even at dye concentrations which produced very poor DNA distributions. Human bone marrow cells were intermediate in their stainability and toxicity response, and acceptable DNA distributions could be obtained at the nontoxic dye concentration of 2.5 muM. Clearly, different cell types differ considerably with respect to their cytotoxic and kinetic responses to Hoechst 33342. In some cases it may not be possible to ensure adequate staining of the cells for flow cytometry without significantly altering their viability and/or proliferative behavior.