Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells

1990 ◽  
Vol 48 (3) ◽  
pp. 912-913
Author(s):  
Jaang J. Wang ◽  
Cheng C. Chen ◽  
Men F. Shaio ◽  
Chia T. Liu ◽  
Chung S. Lee ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cytopathic Effect ◽  
Fetal Bovine Serum ◽  
Virus Particles ◽  
Electron Microscopies ◽  
Embedding Technique ◽  
Fetal Bovine ◽  
Flat Embedding ◽  
20 Nm ◽  
Labeling Method

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.

Fetal bovine serum induces changes in fatty acid composition ofTrypanosoma cruziphosphoinositides

1995 ◽  
Vol 41 (10) ◽  
pp. 951-954 ◽  
Author(s):  
G. Racagni ◽  
M. G. de Lema ◽  
G. Hernández ◽  
E. E. Machado-Domenech
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Trypanosoma Cruzi ◽  
Acid Composition ◽  
Bovine Serum ◽  
Culture Medium ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Fetal Bovine

Fetal bovine serum (FBS) is a necessary constituent of the culture media employed to foster the growth of Trypanosoma cruzi epimastigote forms. In different laboratories, the serum is used at final concentrations of 5 or 10%. We have normally supplemented the complex medium with 10% FBS. Under this condition we have described the fatty acid composition of the total lipids and of the phosphoinositide fractions. Additionally, we have reported the increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. Since further attempts to reproduce these results with 5% FBS in the culture medium were not successful, the effect of the FBS concentration on the fatty acid composition of phospholipids from the T. cruzi epimastigote forms was thoroughly examined. This work showed that when the FBS concentration supplementing the culture medium was reduced from 10 to 5%, the fatty acid composition of the phosphoinositides was altered while the other major phospholipids were not significantly affected. The most relevant result was the decrease in the content of linoleic acid (18:2) and the increase of palmitoleic acid (16:1) in phosphatidylinositol 4,5-bisphosphate. Phosphatidylinositol (PI) and phosphatidylinositol phosphate also exhibited similar changes in the same fatty acids. The C2fatty acid composition of the phosphoinositides, under the same conditions, is also reported here for the first time.Key words: Trypanosoma cruzi, fatty acids, phosphoinositides, fetal bovine serum, phospholipids.

scholarly journals Особливості клітинного циклу мезенхімальних стовбурових клітин з жирової тканини собаки за різних пасажів культивування

2017 ◽  
Vol 19 (78) ◽  
pp. 36-40
Author(s):  
L.V. Kladnytska
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Inverted Microscope ◽  
Proliferative Pool ◽  
Fetal Bovine ◽  
Diploid Cells

The features of the cell cycle of culture of adipose-derived mesenchymal stem cells from the for different cultivating passages were studied. Mesenchymal stem cells were obtained from the adipose tissue of the dog under a laminar flow hood by an explant method in our modification. Cell cultivation was carried out at 37 °C, 100% moisture and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The culture medium was changed 2–3 times per week and the cells were selected by their capacity to attach to the flask surface. When culture flasks became 80% confluence, cells were detached with 0.25% trypsin containing 1 mmol/L EDTA and subsequently replayed at a concentration of 104 cells/cm2 for next passaging. A cells culture of adipose derived mesenchymal stem cells was obtained on the 2nd, 7th and 12th passages. The method of flow cytometry determined the level of aneuploid cells and the distribution in the cell cycle phases. The morphology of cells of different passages was studied using an inverted microscope Axiovert 40. It was investigated that the culture of mesenchymal stem cells from adipose tissue in the 2nd passage contains a significant number of the proliferative pool (S + G2/M) cells and it was 29.51 ± 3.56% of the total number of diploid cells. The number of aneuploid cells was 1.55 ± 0.43%. All cells had fibroblast-like morphology. It was established that in the middle passages (7th) in the culture of mesenchymal stem cells from the adipose tissue of the dog no significant changes were found in the distribution of cells in the phases of the cell cycle. The number of diploid cells of the proliferative pool S + G2/M and the G0/G1 pre-synthetic period remains unchanged. The level of aneuploidy increases only within the tendency. Morphologically, cells had fibroblast-like form. It was determined on 12th passage of cultivation, a significant decrease in the number of cells of the proliferative pool (S+G2/M), which was 18.93 ± 0.66% of the total number of diploid cells compared to the 2nd passage. The number of aneuploid cells increased and it was 3.49 ± 0.38%. Morphologically, separate cells had processes. The indicator of the effect of cells cultivation on the content of diploid cells of the proliferative pool (S+G2/M) in culture is ɳ2x = 70% (P < 0.05). So, first characteristic properties of the aging of the culture of canine adipose-derived mesenchymal stem cells appear on the 12th passage of cultivation.

scholarly journals Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy

2019 ◽  
Vol 20 (5) ◽  
pp. 1053 ◽  
Author(s):  
Tomoyuki Kawase ◽  
Masaki Nagata ◽  
Kazuhiro Okuda ◽  
Takashi Ushiki ◽  
Yoko Fujimoto ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Antigen Expression ◽  
Explant Culture ◽  
The Novel ◽  
Regenerative Therapy ◽  
Platelet Rich Fibrin ◽  
Alp Activity ◽  
Fetal Bovine

In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.

scholarly journals Inhibition of Distinct Proline- or N-Acetylglucosamine-Induced Hyphal Formation Pathways by Proline Analogs in Candida albicans

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Tatsuki Sato ◽  
Hisashi Hoshida ◽  
Rinji Akada
Keyword(s):  
Candida Albicans ◽  
Biofilm Formation ◽  
Culture Medium ◽  
Turning Point ◽  
Fetal Bovine Serum ◽  
Yeast Form ◽  
Induction Condition ◽  
Fetal Bovine ◽  
Virulence Property ◽  
Hyphal Formation

Candida albicans undergoes a yeast-to-hyphal transition that has been recognized as a virulence property as well as a turning point leading to biofilm formation associated with candidiasis. It is known that yeast-to-hyphal transition is induced under complex environmental conditions including temperature (above 35°C), pH (greater than 6.5), CO2, N-acetylglucosamine (GlcNAc), amino acids, RPMI-1640 synthetic culture medium, and blood serum. To identify the hyphal induction factor in the RPMI-1640 medium, we examined each component of RPMI-1640 and established a simple hyphal induction condition, that is, incubation in L-proline solution at 37°C. Incubation in GlcNAc solution alone, which is not contained in RPMI-1640, without any other materials was also identified as another simple hyphal induction condition. To inhibit hyphal formation, proline and GlcNAc analogs were examined. Among the proline analogs used, L-azetidine-2-carboxylic acid (AZC) inhibited hyphal induction under both induction conditions, but L-4-thiazolidinecarboxylic acid (T4C) specifically inhibited proline-induced hyphal formation only, while α-N-methyl-L-proline (mPro) selectively inhibited GlcNAc-induced hyphal formation. Hyphal formation in fetal bovine serum was also inhibited by AZC or T4C together with mPro without affecting the proliferation of yeast form. These results indicate that these proline analogs are ideal inhibitors of yeast-to-hyphal transition in C. albicans.

134 EFFECT OF REPLACING FETAL BOVINE SERUM BY DIFFERENT GROWTH FACTORS IN THE PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS GENERATED BY IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 180
Author(s):  
R. Felmer ◽  
T. Vargas ◽  
R. Sanchez ◽  
M. E. Arias
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Fibroblast Growth Factor 2 ◽  
Bovine Embryos ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Defined Culture

Different culture systems have been studied that support pre-implantation development of bovine embryos up to the blastocyst stage. However, the use of chemically defined culture systems has been less studied. The objective of the present study was to evaluate the effect, in the developmental potential of in vitro-produced bovine embryos, of replacing fetal bovine serum (FBS) by different growth factors in the maturation and embryo culture media. In experiment 1, oocytes collected by aspiration of ovaries from a local slaughterhouse were matured in standard TCM-199 culture medium at 38.5°C, 5% CO2, and saturation humidity. The effect of insulin-like growth factor 1 (100 ng mL–1), epidermal growth factor (10 ng mL–1), and fibroblast growth factor 2 (500 ng mL–1) was evaluated at 24 h by the presence of a polar body after removal of cumulus-oocyte complexes. In experiment 2, oocytes matured in vitro in the presence of FBS were fertilized by co-incubation with commercial sperm (mL) for 18 h in standard fertilization medium (Fert-TALP). The presumptive zygotes were denuded and randomly allocated in a chemically defined culture medium based on KSOM supplemented with polyvinyl alcohol (PVA), fructose, and each of the growth factors listed previously. Undefined cultured medium was based on KSOM supplemented with 5% FBS. Embryos were cultured at 38.5°C in a mixture of gases and saturation humidity. Cleavage and blastocyst rates were recorded on Days 3 and 7, respectively. Analysis of variance was used to test for statistically significant differences between groups (P < 0.05) using Stat Graphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey's test. In experiment 1, a similar maturation rate was observed in all treatments relative to the undefined maturation medium (range = 88–91%). In experiment 2, no differences were observed in the cleavage (79, 87, 85, and 85%) and the blastocyst rates (24, 25, 26, and 30%) for the epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor 2, and FBS treatments, respectively. In conclusion, we demonstrated that maturation of bovine oocytes can be achieved in chemically defined conditions by replacing FBS by each of the growth factors evaluated herein. Furthermore, chemically defined KSOM medium supplemented by any of these growth factors can generate a similar rate of blastocyst than the undefined medium containing FBS. Analyses are under way to evaluate the effect of completely defined culture conditions (maturation and embryo culture) on the pre-implantation development of embryos produced in the presence of these growth factors.

117 Kit ligand enhances growth and nuclear maturation of buffalo oocytes in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 184
Author(s):  
M. N. Islam ◽  
M. H. Alam ◽  
A. Khatun ◽  
M. A. Hashem ◽  
M. Moniruzzaman
Keyword(s):  
Serum Sodium ◽  
Bovine Serum ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Survival Rates ◽  
Sodium Pyruvate ◽  
Antral Follicles ◽  
Kit Ligand ◽  
Fetal Bovine

This study aimed to investigate the effect of Kit ligand (KL), a growth factor that regulates folliculogenesis in mammalian ovaries, on growth of buffalo oocytes in early antral follicles in vitro. Cumulus-oocyte complexes were dissected from early antral follicles (1mm) of slaughtered buffaloes and cultured in Dulbecco’s minimum essential medium supplemented with fetal bovine serum, sodium pyruvate, gentamycin, hypoxanthine, dexamethasone, cysteine, polyvinylpyrolidione, l-ascorbic acid, oestradiol-17β, and androstenedione in a 96-well culture plate at 38.5°C under an atmosphere of 5% CO2 in air for 6 days. The culture medium was supplemented with 0, 50, and 100 ng/mL KL (recombinant human SCF, Cat. No. H8416, R&amp;D Systems, Minneapolis, MN, USA). Sixty oocytes were cultured in each group with 6 replications. In vitro-grown oocytes were cultured for maturation in tissue culture medium-199 supplemented with 5% fetal bovine serum, sodium pyruvate, gentamycin, and 100 ng/mL FSH at 38.5°C for 24h under an atmosphere of 5% CO2 in air. The oocytes were then stained with aceto-orcein and examined under a differential interference contrast microscope. Data were analysed using SAS/STAT version 9.1.3 for Windows (SAS Institute Inc., Cary, NC, USA) by one-way ANOVA and means compared with Tukey’s HSD test. The mean diameter of oocytes measured at the time of seeding on the culture substrate was 100.6±0.4μm (n=180). After 6 days of culture, the diameters of oocytes increased to 110.8±0.5, 114.0±0.5, and 115.0±0.6µm in 0, 50, and 100 ng/mL KL-treated groups, respectively. The survival rates were 60.0±6, 81.2±1.2, and 92.0±4.9% in 0, 50, and 100 ng/mL KL-supplemented oocytes at Day 6. Moreover, KL pretreatment enhanced maturation of buffalo oocytes dose dependently. A small proportion of oocytes (8.4%) treated with 50 ng/mL KL reached the MII stage. This number increased to 25% when oocytes were treated with 100 ng/mL KL. These results show that KL enhances growth, viability, and meiotic progression of buffalo oocytes in vitro.

A combination of bovine serum albumin with insulin–transferrin–sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metalloproteinases

2019 ◽  
Vol 31 (2) ◽  
pp. 333 ◽  
Author(s):  
Ayman Mesalam ◽  
Kyeong-Lim Lee ◽  
Imran Khan ◽  
M. M. R. Chowdhury ◽  
Shimin Zhang ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Sodium Selenite ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Cell Number ◽  
Matrix Metalloproteinase 2 ◽  
Bovine Embryo ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Long Chain

This study investigated the use of bovine serum albumin (BSA) plus insulin–transferrin–sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P&lt;0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P&lt;0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts.

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