A Colchicine,Hypotonic Citrate,Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

A Colchicine, Hypotonic Citrate, Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Flow cytometry: long-term storage of propidium iodide/citrate-stained material.

Single cell suspensions stained by the propidium iodide/hypotonic citrate method for DNA content analysis by flow cytometry can be mixed with an equal amount of 70% alcohol for long-term storage and shipping without introduction of artifacts or loss of fluorescence.     

Flow Cytometry: Long-Term Storage of Propidium Iodide/Citrate-Stained Material

Single cell suspensions stained by the propidium iodide/hypotonic citrate method for DNA content analysis by flow cytometry can be mixed with an equal amount of 70% alcohol for long-term storage and shipping without introduction of artifacts or loss of fluorescence.     

Fast direct method of obtaining metaphase and prometaphase chromosomes from chorion biopsy cells and human embryos during the lst semester of pregnancy]

Samples of chorionic villi and embryonic tissues (brain, brain--sheaths) are thoroughly washed with Hank's solution, immediately subjected to hypotonic treatment (0.9% sodium citrate plus few drops of 0.01% colchicine) 37 degrees C, 30 min, prefixed 20 min with equal amount of standard fixative mixture, twice fixed in standard fixative solution (1 hour, -10 degrees C), hydrated with equal volume of distilled water (5-10 min), dried, macerated directly on the slide with 60% acetic acid. The cell suspension is then evenly spread on the slide surface, dried, postfixed and stained. The method provides sufficient amount of metaphase and prometaphase mitotic plates suitable for differentiating staining in 1.5-2 hours after sampling and might be recommended for routine chromosomal analysis in prenatal diagnosis of inherited diseases during early pregnancy.     

The effects of osmotic stress on human platelets

The effect of osmotic stress on human platelets was investigated at 0, 25, and 37 degrees C. The osmolality of the suspending plasma was decreased by adding water or increased by adding sodium chloride or sucrose. After 5 min, isotonicity was restored by dilution with an excess of isotonic phosphate-buffered saline. After centrifugation, the platelets were resuspended in autologous plasma and then incubated for 1 hr at 37 degrees C before assaying the active transport of 5-hydroxytryptamine (5-HT) and the hypotonic stress response. Anisosmotic conditions had a greater effect on the extent of volume reversal in the hypotonic stress test than on 5-HT uptake. At 25 degrees C, only moderate degrees of hypotonicity (0.25 osmol/kg) or hypertonicity (0.59 osmol/kg) were sufficient to depress the hypotonic stress response. In general, platelets tolerated departures from isotonic conditions better at 0 degree C than at the higher temperatures. Furthermore, at 0 and 25 degrees C approximately equiosmolal concentrations of sucrose and sodium chloride depressed the hypotonic stress response to similar extents, but at 37 degrees C high osmolalities (greater than 2 osmol/kg) were tolerated better when the additive was sucrose than when it was sodium chloride. Platelets shrank when subjected to hyperosmotic conditions, but their discoid shape and the peripheral band of microtubules were maintained.     

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.     

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase alpha activity

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.     

Isolation and characterization of DNA-DNA and DNA-RNA.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.     

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.     

Leukocytes Cultured from Small Inocula of Whole Blood and the Preparation of Metaphase Chromosomes by Treatment with Hypotonic KCL

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO₃, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying method, and staining with 1% natural orcein (G. T. Gurr) in 60% acetic acid. Dehydration and covering completed the preparation. KCI, 0.075 M, has been used advantageously in the above way and for cells cultured by other means from skin and other organs of man and other mammals. Combined advantages of the method are: culture of leukocytes from small volumes of whole blood, with very few failures to obtain mitotic cells; a medium which can be stored frozen in culture vials, and which in a simpler form is usable for long term culture of other cell types; and the use of KCI for hypotonic treatment.     

Intraperitoneal Colchicine and Hypotonic KCl for Enhancement of Abundance and Quality of Meiotic Chromosome Spreads from Hamster Testes

Male meiotic chromosome spreads of the Syrian hamster (Mesocricetus auratus, 2n = 44) were prepared by introducing two modifications into the method of Evans et al. (Cytogenetics, 3: 289-94, 1964). The modifications were pretreatment of the intact animals with colchicine (4 mg/kg injected intraperitoneally) and the use of 0.563% KCl solution to cause swelling of the cells. In animals subjected to colchicine for 1 or 2 hr, there was a markedly increased yield of cells in 6rst meiotic metaphase. This increase was not present in animals subjected for 3 hr, but these animals showed a slight increase of cells in second meiotic metaphase. The use of hypotonic KCl resulted in much sharper chromosome definition than had previously been obtained with 1% sodium citrate solution     

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.     

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.     

Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

Simplified Culture and Chromosome Preparations of Primate Leukocytes

Plasma recovered from 1 ml of primate peripheral blood by centrifugation is planted in a medium consisting of 80% TC-199 and 20% fetal bovine serum, to which 0.125 ml of phytohaemagglutinin/5 ml is added. The pH is adjusted to 7 with 10% NaHCO₂. The mixture is incubated 68 hr, Colcemide to give 1 μg/ml is added, and incubation continued for 4 hr. Following centrifugal separation, the cells are given a hypotonic treatment with 0.75% sodium citrate for 15 min, then centrifuged again and fixed in 3:1 methanol-glacial acetic acid, 3 changes. Tiny drops of the cell suspension are placed on a slide, spread by blowing, and air dried. The preparations are stained with 15% Giemsa solution in methyl alcohol. The method has been successfully used in 256 specimens from 25 different species.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions.

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in DO. Fixation of radiation damage at 37 degrees C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 degrees C than at 20 degrees C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation.