Incubation Of Cell Suspensions Research Articles

Effect of Leu-Enkephalin (Dalargin) on Apoptosis and Necrosis of Leukocytes After Cold Stress

The effect of synthetic leu-enkephalin (dalargin) on apoptosis and necrosis in leukocytes after cold stress on the morphological characteristics of cells and using fluorescent dyes Hoechst 33342 as well as propidium iodide was studied. The degree of cold stress influence depending on the duration of incubation of cell suspension at 0–2 and 37°C on the initiation of apoptosis and necrosis on the morphological characteristics of cells was determined. It has been proven that the ratio of apoptotic cells to necrotic ones was maximal after the use of the regimen as follows: preliminary incubation of leukocytes at 37°C – 15 min, cell exposure at 0–2°C – 15 min and subsequent incubation of leukocytes at 37°C. It has been confirmed that pre-incubation of cells with neuropeptide to cold stress affects the effectiveness of the protective action of dalargin, in particular, contributes to a significant (p ≤ 0.05) reduction in the percentage of leukocytes with morphological signs of apoptosis and necrosis. It was found that a consistent increase in temperature after cold stress to 37°C did not affect the percentage of cells with morphological signs of apoptosis and necrosis. It has been validated that the percentage of leukocytes with the apoptosis and necrosis signs after cold stress was significantly reduced after incubation of leukocytes with dalargin at a concentration of 10–9 mol/l.

Alternative Methods of Evaluation of Antimicrobial Activity of Breast Milk Serum.

Antimicrobial properties are traditionally evaluated by the seeding technique, which is laborious, time-consuming, and rather imprecise. We studied the possibility of using microscopy and spectrophotometry methods for evaluation of cytotoxic activity of breast milk serum against opportunistic microbes. Activity of 50 breast milk samples obtained from healthy women at different lactation stages was tested against opportunistic yeast C. albicans. Microscopy showed that incubation of cell suspension with lactoserum led to destruction of cell walls and cytoplasmic membranes with the formation of vesicular debris that absorbed the dye from the medium. Spectrophotometric measurement of the dye remained in the medium revealed a dose-depended effect of the lactoserum on C. albicans cells and strong inverse correlation between the lactation period and cytotoxic activity of the lactoserum (r=-0.948). These methods can be used in veterinary and food processing for estimation of biological activity of milk.

Micronucleus assay for mouse alveolar Type II and Clara cells

The objective of our study was to develop a micronucleus (MN) assay for detecting genotoxic damage after inhalation exposure in mouse alveolar Type II and Clara cells, potential target cells for lung carcinogens. Ten male C57BL/6J mice were exposed to ethylene oxide (630 mg/m(3)) for 4 hr via inhalation; 10 unexposed mice serving as controls. 72 hr after the exposure, Clara cells and alveolar Type II cells were isolated using two different methods. Method 1 included a 15-min trypsin lavage and a 2-hr incubation of cell suspension. Method 2 involved a 30-min trypsin lavage, Percoll gradient centrifugation, and a 48-hr incubation for cell attachment. Nitro blue tetrazolium (NBT) -staining was applied to distinguish Clara cells. The frequency of micronuclei (MNi) was scored in NBT-negative cells (defined as Type II cells in Method 2) and NBT-positive cells (Clara cells). To detect possible differences between the techniques, MNi in Clara cells were analyzed from samples prepared by both methods. With Method 2, a clear increase in the mean frequency of micronucleated cells was seen in the exposed mice as compared with the controls, for both alveolar Type II and Clara cells. However, no significant increase in MN frequency was seen in Clara cells analyzed from samples prepared by Method 1. Based on our findings, mouse alveolar Type II and Clara cells seem to be suitable for MN analysis in studies aimed at identifying genotoxic lung carcinogens. Both alveolar Type II and Clara cells can be isolated using Method 2.

DNA synthesis during male meiotic prophase in the rat.

DNA synthesis in different stages of the male meiotic prophase of the rat was studied by autoradiography after incubation of small segments of isolated seminiferous tubules with tritiated thymidine. Compared to incubation of cell suspensions, this technique gives the advantage that the physiological contacts between different cells are preserved, which diminishes the damage of the cells and their DNA. In addition, the recognition accuracy of the meiotic stages is increased as the morphology of the developing acrosomes can be used as an additional criterium for identification of the stages of the spermatogenic epithelial cycle. Highest synthetic rate was found in the mid-pachytene spermatocytes, while moderate or low rate was observed in leptotene, zygotene, early pachytene, late pachytene and diplotene stages.

Migration pattern, morphology and viability of cells suspended in or sealed with fibrin glue: A histomorphologic study

Introduction: We studied the migration pattern, morphology and viability of cells suspended in five different fibrin glues. Besides this, the behaviour of chondrocytes seeded on porous matrices comprising different collagen types sealed with fibrin glue was investigated. Material and methods: In an experiment A, cell suspension (0.5 × 10 6 cells) was incubated with different fibrin glues. Experiment B was set up to evaluate chondrocytes migration either through a collagen I/III (Chondro-Gide ®, Geistlich Biomaterials, Switzerland) or collagen II matrix sealed with different fibrin glues in a perfusion chamber system. Analysis were performed by lightmicroscopy (Mayer's hematoxylin-eosin; Masson–Goldner; TUNEL test) and by transmission and scanning electron microscopy. All fibrin glues were measured for TGF-β 1 and 2 with a specific ELISA. Results: After incubation of cell suspension in autologous fibrin glue, the morphology of cells is chondrocyte-like. Spindly, process-bearing cells were seen in commercial fibrin glue. Cells suspended in commercial fibrin glue revealed a significant higher percentage of TUNEL positive cells compared to fibrin tissue adhesives mixed with autologous serum ( p = 0.006). The TGF-β 1 and 2 concentration was significantly higher in partial autologous fibrin sealant (PAF) compared to their commercial counterparts ( p = 0.001). Cells seeded on the collagen I/III matrix retained their chondrocytic morphology, while in the type II collagen matrix the chondrocytes displayed a fibroblastic phenotype. The ratio of TUNEL positive cells for the collagen I/III matrix was significantly surpassed by the values, when a collagen II matrix was used ( p = 0.008). No ingrowth of cells was seen in any of the experimental conditions. Conclusion: Partial autologous fibrin glue and collagen I/III matrices are favourable in respect to migration pattern, morphology and viability, but definitive conclusions can only be drawn after in vivo studies. This will be addressed in future animal studies.

IMMUNOMAGNETIC CELL ENRICHMENT DETECTS MORE DISSEMINATED CANCER CELLS THAN IMMUNOCYTOCHEMISTRY IN VITRO

We describe a method to improve tumor cell detection compared to currently available immunocytochemical methods by using immunomagnetic cell enrichment. Two different methods of immunomagnetic cell enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry. One method was positive selection of epithelial cells from mononuclear cells with the antiepithelial antibody BER-EP4 and the other was depletion of mononuclear cells with the antileukocyte antibody CD45. Mononuclear cells were isolated from peripheral blood by density centrifugation and various numbers of tumor cells were added. The 5 different cell lines from urological malignancies used in the study were DU-145, RT-4, CAKI-2, KTCTL-2 and KTCTL-30. Following incubation of cell suspensions with the beads, cell separation was performed in a magnetic field. After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed. A total of 112 experiments were completed and negative controls were obtained. The number of tumor cells detected by positive selection and depletion was significantly higher than by immunocytochemistry (p <0.001). The median enrichment factor and tumor cell recovery rate for positive selection and depletion were 15.3 and 61.2%, and 13.0 and 57.3%, respectively (not significant). With less than 1 tumor cell suspended in 106 mononuclear cells, the probability of tumor cell detection was 23% for immunocytochemistry alone and 93.3% for both enrichment methods (p <0.01). No false-positive results were observed. Compared to immunocytochemistry, immunomagnetic cell enrichment significantly improves the sensitivity of detection of epithelial cells added to mononuclear cells. Both methods of enrichment were equally effective and may be important for clinical practice in the future.

Effect of bile on the $\beta$-galactosidase activity of dairy propionibacteria

In the present study, the effect of bile on the growth, cell permeabilization and β-galac- tosidase activity of dairy propionibacteria was determined. Two groups of strains with different behavior in media with oxgall (dehydrated fresh bile) could be observed, and were named bile-tol- erant and non bile-tolerant groups. The β-galactosidase activity of bile-tolerant strains increased during the growth in media containing 0.15 and 0.30% oxgall. The incubation of cell suspensions in phosphate buffer with oxgall increased the β-galactosidase activity of all the studied strains. They were permeabilized by bile, permitting more substrate to enter the cells to be hydrolyzed by β-galactosi- dase. A higher specific activity was observed in cell-free extracts obtained from cultures in media with oxgall, indicating increased enzyme synthesis in the presence of bile. The enzyme activity was also increased by sodium taurocholate and sodium chloride. These results suggest that bile-tolerant strains of propionibacteria can provide meaningful β-galactosidase activity by permeabilization of the cells and increased enzyme synthesis during growth in the gut. The non bile-tolerant strains did not grow in media with bile. They were permeabilized, but their β-galactosidase was inactivated when they were exposed for a long time to bile in the growth media. Thus, their contribution to the β-galactosidase activity in the gut does not appear to be significant. Propionibacteria / bile tolerance / β-galactosidase

Communications: Phenotypic Variability in Pedigreed Families of Goldfish Revealed by Alloantisera and Agglutination Assays

Techniques of demonstrated usefulness for observing genetic variability at the major histocompatibility complex (MHC) in avian and mammalian systems were evaluated for their potential utility in piscine systems by using goldfish Carassius auratus as a model. Three approaches to generating antisera and two methods of observing the results of hemagglutination assays were evaluated. Seven full-sib families were produced, and red blood cells (RBCs) of goldfish family members were screened for reactivity with a panel of absorbed antisera consisting of fish anti-fish, MHC-specific chicken anti-chicken, and chicken anti-fish sera. The pattern of presence or absence of agglutination after incubation of cell suspensions with the respective reagents in the panel of antisera was regarded as the phenotype of the individual tested. Fifty different hemagglutination phenotypes were observed within and between families. The large numbers of phenotypes observed within particular families may indicate (1) the need for additional absorptions in the preparation of antisera useful for observing variability of the locus or loci of issue, (2) segregation of additional sets of MHC or other haplotypes in the tetraploid goldfish, or (3) segregates of non-MHC loci reacted with the antisera produced. Results of this study suggest that the development of immune antisera and use of hemagglutination assays eventually might be used to characterize genetic variability at the MHC in fish populations.

Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya.

The biosynthesis of organofluorine compounds by Streptomyces cattleya NRRL 8057 was examined using 19F NMR spectroscopy. The organism produced 1.2 mM fluoroacetate and 0.5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two fluorometabolites in almost equal proportions. As the harvest age increased, the proportion of fluoroacetate to 4-fluorothreonine formed by cell suspensions rose progressively so that 16-d-old cells showed a ratio of 76:26 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate at pH 6.0 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable at pH 7.5. Fluoroacetate and 4-fluorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological defluorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of spheroplasts from the strict anaerobe Selenomonas ruminantium

A method for the preparation of spheroplasts from Selenomonas ruminantium is described. The method is based on the lysozyme/EDTA method developed by Kaback and collaborators. The most important modification introduced consisted in the substitution of nitrilotriacetic acid for EDTA. The final yield of spheroplasts was ≈80% of the original whole cell suspension. However, to achieve this yield, the inclusion of n-decanoic acid and higher yeast extract levels in the growth medium, as well as the substitution of mutanolysin for lysozyme and of nitrilotriacetic acid for EDTA during the incubation of cell suspensions at lower temperatures, were also all found necessary. Right-side-out vesicles were obtained from these spheroplasts according to the original method.

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: Differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37°C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[ 3H]Phe for up to 30 min at 37°C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 μM. 50% inhibition was observed at 1–10 μM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 μM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.

Effect of Calcium, Sodium and Isoproterenol on Renin Secretion From Disaggregated Rat Renal Cortical Cells

A disaggregated cell system prepared from rat renal cortices has been developed to study the regulation of renin release in vitro. Cell suspensions were prepared by incubating minced renal cortical tissue in collagenase. The digested tissue was filtered sequentially through graded Teflon meshes of 125 and 44 mu, respectively. This preparation contained less than 2% intact glomeruli or tubules. Incubation of cell suspensions with the beta-adrenergic agonist L-isoproterenol (ISO) significantly increased the activity of renin released into the incubation media. Peak levels of renin activity were detected 10 to 15 min after the addition of ISO. The apparent ED50 for stimulation of renin release by ISO was 6 X 10(-8)M and the response was antagonized by the beta-adrenergic antagonist dl-propranolol. The spontaneous release of renin was also suppressed by increasing the concentration of extracellular calcium, whereas sodium ions had no effect on this process. These data have demonstrated that disaggregated renal cortical cell models are useful for studying the in vitro effects of pharmacologic agents and ions on renin secretion.

Ultrastructural localization of the transferrin receptor and transferrin on marrow cell surfaces

The monoclonal antibody OKT9 (a known transferrin receptor antibody) and a monoclonal antibody to transferrin (ATfn) were used to localize the transferrin receptor and transferrin on marrow cells. After incubation of cell suspensions with the antibody, the cells were reacted with an affinity purified Fab fragment of goat anti-mouse IgG conjugated to horseradish peroxidase (GAM-HRP), which was in turn visualized by reaction with 3,3'-diaminobenzidine (DAB). Erythroblast cell surfaces stained intensely with OKT9-GAM-HRP-DAB, weaker staining was observed on reticulocyte surfaces, whereas erythrocyte surfaces lacked staining. Staining was present on surface caveolae, which often contained endogenous ferritin particles. Moderate to strong OKT9 staining was observed on less than 10% of presumed lymphoid cells. Monocytes, macrophages, promyelocytes, granulocytes, megakaryocytes and platelets consistently lacked OKT9 staining. ATfn-GAM-HRP-DAB staining of erythroid cells was similar to that observed with OKT9 staining; however, in contrast to the latter staining, ATfn stained the surfaces of megakaryocytes, platelets, monocytes and most lymphocytes. Promyelocytes stained weakly, whereas late granulocytes lacked staining. These results indicate that the T9 transferrin receptor (1) is largely confined to erythroid cells in marrow, (2) is diffusely distributed on the surface of early erythroid cells, (3) decreases with cell maturation, and (4) is lost when haemoglobin synthesis is completed. Transferrin appears in a similar distribution on erythroid cell surfaces but also appears to bind to some cell surfaces lacking the T9 receptor.

Regulation of tyrosine hydroxylase activity in retinal cell suspensions: Effects of potassium and 8-bromo cyclic AMP

Tyrosine hydroxylase (TH) contained in dopamine (DA) neurons of rat retina is activated in vivo as a consequence of photic stimulation. Experiments were conducted to test the effects of changes of membrane potential and of cyclic AMP-dependent protein phosphorylation on TH activity of these retinal neurons. Retinas were dissociated into suspensions of apparently viable cells to test the direct effects of pharmacological manipulations of TH activity in the absence of trans-synaptic influences. To test the effects of changes of membrane potential on TH activity we examined the effects of a depolarizing agent, potassium. Incubation of cell suspensions in Ringer's solution containing a depolarizing concentration of K + (52 mM) resulted in a significant increase of TH activity, suggesting that membrane depolarization may trigger a series of molecular events that leads to TH activation. Incubation of cell suspensions in the presence of 8-bromo cyclic AMP, a cyclic AMP analog that is known to activate cyclic AMP-dependent processes following extra-cellular application, resulted in a significant activation of TH that was comparable to that produced in vitro by cyclic AMP-dependent protein phosphorylation. These data support the hypothesis that membrane potential plays a role in the regulation of TH activity, and indicate that cyclic AMP-dependent phosphorylation can activate retinal TH in situ . The apparent viability of the retinal cells in suspension suggests that this preparation may be useful for studying synaptic regulatory mechanisms.

The application of particle size analysis to the aggregation of Chinese hamster cells by concanavalin A

A Coulter counter and pulse height analyser has been used to study the aggregation of Chinese hamster cells in the presence of concanavalin A (ConA). After incubation of cell suspensions with ConA under standard conditions of agitation equilibrium aggregate size distribution curves are obtained which show that there is a greater proportion of larger aggregates formed than during aggregation in the absence of the lectin. At equilibrium there is a significant population of single cells. The mean number of cells per aggregate increases with temperature and goes through a maximum as a function of ConA concentration. The kinetics of the aggregation process exhibit features consistent with a bridging mechanism of aggregation mediated by the lectin and leading to an enhanced cellular adhesiveness but his process is superimposed on aggregation arising as a result of the inherent adhesiveness of the cells.

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.

Regulation of hydrogenase in Rhizobium japonicum

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.

The inhibition by 6-diazo-5-oxo-l-norleucine of glutamine catabolism of the cultured human lymphoblast.

The rapid catabolism of glutamine by the cultured human lymphoblast line WI-L2 can be inhibited greater than 95% by incubation of cell suspensions with 6-diazo-5-oxo-L-norleucine (DON). The inhibition persists for at least four hours after removal of DON from the cell suspension. The exposure of cells to DON ihibits over 95% of the glutaminase activity measured in lysates in the presence of either phosphate or maleate. Similarly, gamma-glutamyl transpeptidase, assayed with gamma-glutamyl-p-nitroanilide as substrate and glycyglycine as acceptor, is inhibited over 90%. DON-treated and control cells accumulated radioactive material from suspensions containing [14C]-L-glutamine at similar initial rates; the radioactive material accumulated by the DON-treated cells is all recoverable as glutamine while the radioactive material accumulated by untreated cells is principally recovered as glutamate.

Métabolisme de l'acide indolyl-3-acétique dans les cultures de cellules d'Acer pseudoplatanus cultivés in vitro

Incubation of cell suspensions of sycamore (Acer pseudoplatanus) with β-indoyl-3-acetic acid (IAA) first led to the formation of IAA-glycosides, then to that of IAA-aspartate. Great differences are observed between the kinetics of IAA transformed by two distinct strains: one, auxin dependent (S), the other, auxin independent (MB). Other degradation products are only found in the culture medium. The localization of IAA-degrading systems in the cell wall is postulated. The auxin requirement of the S strain is discussed.

Chilling of somatic cells unnecessary for Sendai virus-induced heterokaryon formation

Incubation of cell suspensions or monolayers at 4 degrees C for 20 min after addition of beta-propiolactone-inactivated Sendai virus did not enhance heterokaryon formation as compared to parallel cell cultures incubated at 37 degrees C immediately after the addition of Sendai virus. These findings show that the routine chilling of somatic cells in fusion experiments can be omitted.