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 rapid single step staining technique for DNA analysis by flow microfluorimetry

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.     

Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data

In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure. Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intentsity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 muM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.     

Viability assessment of honey bee, Apis mellifera, sperm using dual fluorescent staining

Since the development of instrumental insemination of honey bee (Apis mellifera) queens in the 1930s, there has been interest in the evaluation and in vitro storage of semen. Several fluorescent stains, when used in combination, have been effectively used to assess sperm viability in mammalian and avian species. Our objectives were to test two combinations of living:dead fluorescent stains, SYBR-14 with propidium iodide (PI), or Calcein-AM with PI, and validate the use of these probes with honey bee sperm. SYBR-14 is a nuclear stain producing green fluorescence of the DNA in living sperm, Calcein-AM is a membrane-permeant esterase substrate staining entire sperm green, and PI is a traditional dead cell stain giving a contrasting red color. Both living stains fluoresced bee sperm, but the SYBR-14:PI produced a clearer distinction between the living and dead sperm. A graduated series of known living:dead sperm proportions was used to validate the accuracy of the stains for determining sperm viability in honey bees.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

Flow cytometry as an estimation tool for honey bee sperm viability

Flow cytometry is a method to conduct a multiparameter analysis of cells suspended in liquid and passing through a laser beam. Analyses of human and other mammal sperm using this method have already been performed but its application for insect semen is still the subject of investigation. Semen isolated from honey bee Apis mellifera seminal vesicles was dyed using SYBR-14 and propidium iodide (PI). The fluorescence of the SYBR-14 stained cells was analyzed in a green fluorescence channel (FL-1), while the PI fluorescence was analyzed in a red fluorescence channel (FL-3). Living and dead cell populations were separated using a density dot plot and the percentage of each in the sample was calculated. Flow cytometry seems to be an effective tool for assessing the viability of honey bee semen, solving the problems of distinguishing and counting the double-stained cells.     

Qualitative aspects of sperm stock in males and females from Eupelmus orientalis and Dinarmus basalis (Hymenoptera: Chalcidoidea) as revealed by dual fluorescence

Abstract The quality of a sperm population can be characterized physiologically and its fecundity predicted by its viable : non-viable sperm ratio. To improve the knowledge of reproductive strategies in two ectoparasitoid hymenopteran species, Eupelmus orientalis Crawford (Hymenoptera: Eupelmidae) and Dinarmus basalis Rondani (Hymenoptera: Pteromalidae) , the assessment of sperm viability using the dual fluorescence staining procedure SYBR-14 : propidium iodide was developed. The aim of the study was to provide a comparative test in vitro applicable to both sexes to study the evolution of sperm quality at various stages of the reproductive processes. The reliability of propidium iodide to detect non-viable sperm (stained in red) was confirmed in both species on the basis of two stress tests (ethanol and Triton X-100) but our study also revealed that propidium iodide concentrations must be adequately adjusted for each single species. This experiment also demonstrated the physiological heterogeneity of sperm populations in E. orientalis and D. basalis males and females. In both species, 40% of the sperm in the seminal vesicles was found to be non-viable. By contrast with E. orientalis, the populations of non-viable sperm estimated from the seminal vesicles of D. basalis were found to be strongly different from those observed in the spermatheca. From the present results, the population of viable sperm detected in the spermatheca of females from both species proved a reliable predictor of fertilization achieved in ovipositing females.     

Flow microfluorometric analysis of cellular DNA: Critical comparison of mithramycin and propidium iodide

Mithramycin and propidium iodide were used to stain HeLa cells, human lymphoma cells, and phytohemagglutinin-stimulated lymphocytes for flow microfluorometric analysis of cellular DNA. The stains provided similar estimates for the proliferative fraction of the populations. However, significant differences in the relative fluorescent intensity were demonstrated in the three cell populations. Fluorescent intensity of HeLa and lymphoma cells stained with mithramycin was higher than matched propidium iodide-stained cells. Normal lymphocytes showed greater fluorescent intensity when stained with propidium iodide. Differences in the staining behavior of these two dyes may prove to be highly informative probes of chromatin structural differences.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining

A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.     

Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single-wavelength fluorescence microscopy.

The purpose of this study was to use fluorescence microscopy to determine the viability and acrosome status of fresh and frozen-thawed human spermatozoa. Sperm cells were stained with the viability stains Hoechst 33258 (H33258) alone, or propidium iodide (PI) alone, and PI in combination with FITC-conjugated Pisum sativum agglutinin (PSA). The PSA stains the acrosome contents of permeabilized acrosome-intact sperm. Viability by fluorescence microscopy was compared to conventional eosin nigrosin staining. The overall viability using H33258 was not significantly different from that using PI. Therefore, PI was used in combination with PSA for simultaneous measurement of viability and acrosome status at the same excitation wavelength (488 nm). By combining PI and PSA, four subgroups of cells could be detected: group I, PI-neg/PSA-neg--viable, physiologic acrosome reacted (AR); group II, PI-neg/PSA-pos--viable, non-AR; group III, PI-pos/PSA-neg--nonviable, non-AR; group IV, PI-pos/PSA-neg--nonviable, degenerative AR. The postthaw sperm exhibited a significantly greater percent of sperm that were acrosome reacted (both viable and degenerative) (groups I and IV) than the fresh semen. We conclude that frozen-thawed sperm may undergo premature break-down of the acrosome prior to interaction with the oocyte, thus explaining the reduced fertility potential of cryopreserved semen.     

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 micrograms/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.     

A stable propidium iodide staining procedure for flow cytometry

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.     

Apoptotic Effect of Melittin Purified from Iranian Honey Bee Venom on Human Cervical Cancer HeLa Cell Line

Melittin, an amphipathic 26-residue peptide, is the main component of honey bee venom. Studies have been demonstrated that melittin has an inhibitory effect on proliferation of cancer cells. However, the precise mechanism of action is not completely understood. In the present study we have shown that purified melittin from Iranian honey bee venom shows anti-cancer effects on human cervical cancer cell line through induction of apoptosis. The venom was collected from Iranian honey bee (Apis mellifera meda) and melittin isolated using reversed phase HPLC. Biological activity of melittin was analyzed by hemolytic test on human red blood cells. In order to investigate whether melittin inhibits proliferation of cervical cancer cells, the viability of the melittin treated HeLa cell line was measured via MTT assay. Finally, cell death analysis was performed using Propidum iodide and Annexin V-FITC dual staining. The results showed that the half hemolytic concentration (HD50) induced by mellitin was 0.5 µg/ml in free FBS solution. IC50 obtained after 12 h at 1.8 µg/ml by MTT assay. According to flow cytometric analysis, melittin induced apoptosis at concentrations more than 1 µg/ml. These results suggest that melittin induces apoptotic cell death in cervical cancerous cells as observed by flow cytometric assay. It is concluded that melittin could be regarded as a potential candidate in future studies to discovery of new anticancer agents.     

Cryptosporidium parvum sporozoite staining by propidium iodide.

Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlorite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts' DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments.     

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.