Measurement of efflux from G1-phase in a growth factor dependent cell line

In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the Gl-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.     

A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

A new technique to identify hybrid myotubes in vitro without culture fixation.

Fluorescent latex microspheres (FLMs) were used to label myoblasts and to permit the observation of hybrid myotubes before culture fixation. This type of labeling did not affect survival, development, or fusion of these cells. The FLMs were retained for several weeks. Labeled mouse myoblasts were co-cultured with unlabeled rat myoblasts to verify whether the marker was released and spread from labeled to unlabeled cells. The nuclear stain Hoechst 33258 was used to distinguish the myoblasts from both species and permitted the demonstration that there was virtually no re-uptake. Hybrid myotubes were also obtained by co-culturing mouse myoblasts containing rhodamine FLMs and rat myoblasts containing green FLMs. These mixed cultures were observed repeatedly with a fluorescent microscope without any cytotoxic effect. Several myotubes were observed before fixation of the cultures to contain both types of fluorescent labels. Subsequent fixation and staining with Hoechst dye confirmed that these myotubes were hybrids.     

Note from the Biological Stain Commission: Certification of Wright Stain Solution

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting (FACS)

Studies using molecular markers have revealed that planarians possess a highly organized brain. Here we separated brain neurons from dissociated planarian head cells by fluorescence activated cell sorting (FACS), and characterized them by single cell PCR analysis and cell culture. Dissociated cells were labeled with three different fluorescent dyes, Hoechst 33258, Merocyanine 540, and Propidium Iodide (PI), and fractioned by FACS. Interestingly, we have succeeded in identifying a cell fraction specific to the head, which we have named the head-abundant cell fraction (HAC). Most of the HAC expressed neuron-specific genes and proteins. When they were cultured in vitro, they showed an ability to extend neurites on several types of extracellular matrices (ECMs), and, depending on the ECM type used, presented a high level of plasticity in morphology and gene expression.     

Vital DNA staining and cell sorting by flow microfluorometry

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.     

Programmed cell death induced by cadmium stress and/or boron deprivation in tobacco (Nicotiana tabacum L.) cultivar Bright Yellow 2 cells

Cadmium (Cd) is one of the most toxic and widespread heavy metal pollutants in soil. As an essential mineral nutrient, boron (B) plays critical roles in physiological processes of plants. In the present study, programmed cell death (PCD) induced by Cd stress and/or B deprivation was assessed and the underlying mechanisms were clarified in suspension-cultured Nicotiana tabacum L. cultivar Bright Yellow 2 (TBY-2) cells. The PCD in TBY-2 cells was analyzed by Hoechst 33258 staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and then, expression analysis of PCD-related genes was performed using quantitative real-time polymerase chain reaction (qPCR) assays. The production of reactive oxygen species (ROS) was determined using fluorescence microscopy of 2′,7′-dichlorofluorescein diacetate–labeled cells. The levels of lipid peroxides were quantified by the thiobarbituric acid–reactive substances (TBARS) method. Cadmium stress and/or B deprivation treatments induced PCD that was characterized by a significant increase in the percentage of cells stained with Hoechst 33258 or TUNEL-positive cells, and upregulation or downregulation of the expression of PCD-related genes. Treatments with Cd stress and/or B deprivation increased ROS production and the level of lipid peroxides compared to those of the control group. These data showed that in TBY-2 cells Cd stress and/or B deprivation activated ROS signaling pathways, leading to gene expression that was connected with the PCD process.

     

Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Elimination of mycoplasma contaminants from cell cultures with animal serum

Repeated treatment with guinea pig or rabbit serum, but not with human serum, was found to eliminate mycoplasma contaminants from mammalian cell cultures as judged by staining with the fluorescent dye Hoechst 33258. Following treatment with rabbit serum and several passages, M. hyorhinis could not be detected by staining, isolation on agar, or specific immunofluorescence in a human prostate carcinoma cell line heavily contaminated with this organism. There was no evidence for the involvement of antimycoplasma antibodies in the bactericidal activity of rabbit serum. Mycoplasmacidal activity of rabbit serum was associated with a heat-labile component(s) which could be inactivated by incubation of the serum with goat antirabbit complement component C3.     

A fast and robust quantitative time-lapse assay for cell migration

We describe a simple and widely applicable method to measure cell migration in time-lapse sequences of fluorescently labeled cells in culture. Briefly, binarized cell images obtained after thresholding were cumulatively projected, and the covered areas were measured. This procedure determines the time course of the track area successively covered by the cell population. Under conditions where cell growth is negligible, a robust index of cell motility is derived from normalized plots for the displacement of cells over time. We applied this method to quantitatively examine the migration of B35 neuroblastoma cells transiently expressing GFP and to C6 glioma cells after staining with Hoechst 33258. This sensitive assay detected the influence of agents which inhibit actin polymerization (cytochalasin B) or interfere with the maintenance of cell polarity (methyl-beta-cyclodextrin) on cell migration. Thus, this assay is a versatile tool to measure quickly the migration of different cell types using different labeling strategies.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Human skeletal muscle stem cell antiinflammatory activity ameliorates clinical outcome in amyotrophic lateral sclerosis models

Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle-derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm-SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.     

Genetics of cell fusion: human chromosome 10 assignment of a gene (FUSE) that promotes polykaryocyte formation.

FUSE, a human gene which promotes polykaryocyte formation, has been identified and examined in cocultivation assays between rat XC cells and human-mouse hybrids retaining different combinations of human chromosomes. Polykaryocyte formation was never detected when parental cells of hybrids were cocultivated with XC cells. Somatic genetic synteny analysis employing different hybrid sets demonstrated that FUSE was coexpressed with the chromosome 10 markers glutamate oxaloacetate transaminase (GOTs) and an external membrane protein (EMP-130). Cytogenetic analysis confirmed this assignment to human chromosome 10. FUSE was expressed by hybrids made with both human leukocytes and fibroblasts from several individuals, indicating the gene is found in different tissues and may be ubiquitous. Only XC cells were involved in polykaryocyte formation as demonstrated by 33258 Hoechst staining and the absence of heteropolymers between rat and cell hybrid multimeric enzymes. Evidence suggests that the gene FUSE produces a nondiffusible and noninfectious product that is associated with the human-mouse hybrid surface.     

Quenching of Hoechst 33258 fluorescence in erythroid precursors.

Quenching of the fluorescence of DNA-bound Hoechst 33258 in erythroid precursors was studied by flow cytometry and cytochemistry. This quenching artifact may affect the measurement of ploidy in specific cases. The bone marrow cells of two patients with hemolytic disease and active erythropoiesis contained subpopulations of cells with an apparent hypodiploid DNA content as measured by flow cytometry of paraformaldehyde-fixed cells stained with Hoechst 33258. No aneuploidy was detected in either of the two cases when cells were stained with mithramycin or 7-aminoactinomycin D. Cells exhibiting reduced Hoechst 33258 fluorescence expressed glycophorin A and low amounts of CD36, and were therefore erythroid precursors. In one case studied, the number of cells with reduced Hoechst 33258 fluorescence and glycophorin A expressed agreed well with the number of cells containing nuclear hemoglobin. In the other case, hemoglobin was present in a significant proportion of nucleated cells. Calculated values for the efficiency of resonance energy transfer from Hoechst 33258 to hemoglobin were in accordance with the observed levels of quenching (approximately 10%). However, the results could also be explained by hemoglobin reabsorption of Hoechst 33258 fluorescence. Nuclei stained with Hoechst 33258 showed uniform fluorescence, probably due to extraction of hemoglobin during the isolation procedure.     

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.     

Fluorescence spectra of Hoechst 33258 bound to chromatin

Fluorescence spectra of Hoechst 33258 bound to rat thymocytes were measured by flow cytometry. At low dye concentrations (less than or equal to 2 micrograms/ml) the fluorescence maximum was situated at 460 nm irrespective of solvent composition. With higher dye concentrations the fluorescence maximum was shifted upwards, the intensity decreased and the width of the fluorescence peak increased. Linear combinations of a spectrum obtained at a low dye concentration (0.5 microgram/ml, type 1 binding) and one obtained at a high dye concentration (42.4 micrograms/ml, type 2 binding) failed to reproduce spectra measured at intermediate dye concentrations (0.15 M NaCl). Hence, Hoechst 33258 forms at least three different fluorescing complexes with DNA in chromatin. The shift in the fluorescence maximum of the Hoechst 33258/chromatin complex towards higher wavelengths decreased with ionic strength. 25% ethanol in the 0.15 M NaCl staining buffer reduced the wavelength shift at high dye concentrations, indicating that the strength of type 2 binding depends on DNA conformation in addition to ionic strength. The fluorescence spectrum was independent of whether DNA in chromatin was complexed with histones or not. However, histone-depleted thymocytes fluoresced more intensely than cells in which DNA was complexed with histones, the difference being greater at low concentrations of Hoechst 33258. Hence, type 2 binding to DNA in chromatin appears to be less restricted by histones than type 1 binding.     

Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix

The interactions of DNA-binding dyes (Hoechst 33258, DAPI, acridine orange) and DiBAC₄(3) with hyperthermophilic archaeon Aeropyrum pernix cells were investigated by the combination of calorimetric, spectroscopic and microscopic techniques. All of the dyes, studied here, affect the thermal stability of DNA in vivo and in vitro. Hoechst 33258 is highly DNA-specific probe, which does not affect the thermal transitions of other cellular components as can be detected by differential scanning calorimetry (DSC). Due to this unique property, it can be used as a potential DNA marker for in vivo DSC studies. The localization of the dyes in the cells and viability assay was revealed by fluorescence microscopy. Hoechst 33258, DAPI and acridine orange did not distinguish between viable and non-viable cells of Aeropyrum pernix. Only with the commercially available Live/Dead BacLight^TM kit we were able to discriminate viable and non-viable Aeropyrum pernix cells.     

A cytochemical and immunocytochemical study of DNA distribution in spermatid nuclei of mouse,rabbit, and bull

Summary DNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species-specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nuclei contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst-fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species-specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements.