The Use of In Vitro Cultured Lymphocytes for Tracing Mutagenic Activities of Chemicals

1987 ◽  
Vol 14 (3) ◽  
pp. 168-171
Author(s):  
J. Clausen ◽  
S.A. Nielsen
Keyword(s):  
Dna Damage ◽  
Peripheral Blood ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Significant Rise ◽  
Time Dependent ◽  
Alkaline Elution ◽  
Rpmi Medium ◽  
Repair Enzymes

Lymphocytes from normal, non-smoking human individuals not taking drugs were isolated from the peripheral blood by means of the lymphoprep method. The cells were cultured in RPMI medium with 10% fetal calf serum and stimulated with Phytohemagglutinin. A mutagen such as 3-methylcholanthrene was added for varying periods of time. Then the subspecies of DNA, i.e. double and single stranded DNA (ds-DNA and ss-DNA), were separated by the alkaline elution technique and quantitated by fluorimetric estimation. The mutagen induced a significant rise in the level of ss-DNA, but no changes in ds-DNA could be traced. The time-dependent changes increased for at least four days of exposure, indicating that the repair enzymes were not able to compensate for the DNA damage.

scholarly journals Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

2011 ◽  
Vol 75 (3) ◽  
pp. 429-433 ◽  
Author(s):  
F.G. Leivas ◽  
D.S. Brum ◽  
S.S. Fialho ◽  
W.P. Saliba ◽  
M.T.T. Alvim ◽  
...  
Keyword(s):  
Fetal Calf Serum ◽  
Bos Taurus ◽  
Calf Serum ◽  
In Vitro Production ◽  
Bos Taurus Indicus ◽  
Vitro Production

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Jana Holmar ◽  
Heidi Noels ◽  
Joachim Jankowski ◽  
Setareh Orth-Alampour
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Vascular Smooth Muscle Cells ◽  
Incubation Time ◽  
Fetal Calf Serum ◽  
Ex Vivo ◽  
Calf Serum

Abstract Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

scholarly journals Nuclear Damage in Peripheral Erythrocytes of Cyprinus Carpio Exposed to Binary Mixture of Pesticides

2019 ◽  
Vol 2 (1) ◽  
pp. 39-47
Author(s):  
Faiza Ambreen ◽  
Muhammad Javed
Keyword(s):  
Dna Damage ◽  
Cyprinus Carpio ◽  
Peripheral Blood ◽  
Damage Index ◽  
Tail Length ◽  
Time Dependent ◽  
Lethal Concentration ◽  
Static System ◽  
Genetic Damage ◽  
Long Duration

The present study was undertaken to examine the DNA damage in peripheral blood erythrocytes of Cyprinus carpio under the binary exposure of bifenthrin and chlorpyrifos by using single cell gel electrophoresis (SCGE). Limited efforts have been made to study the genotoxic effect for long duration period. Therefore, the present investigation was aimed to assess the genotoxicity of pesticide mixture to the freshwater carp, Cyprinus carpio at sub-lethal concentration exposure (33% LC50). At first 96-hr LC50 value of pesticide, the mixture was determined for Cyprinus carpio in a static system and then sub-lethal concentration was calculated and fish was exposed to this sub-lethal concentration of the mixture in glass aquaria for 70 days (five fortnights) at constant laboratory conditions. Peripheral blood erythrocytes were taken on a fortnightly basis for the time-dependent DNA damage assessment in-terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets. Concentration-dependent increase in the percentage of DNA damaged cells were observed up to a 4th fortnight, followed by a slight decrease in the 5th fortnight. Similarly, statistically significant time-dependent DNA damage was observed in terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets in treated fish (at 33% of LC50) as compared to control groups. The results supported the use of SCGE for evaluating the toxicity of pollutants which may be used as part of environmental monitoring programs.

scholarly journals In vitro antibacterial activity of omeprazole and its selectivity for Helicobacter spp. are dependent on incubation conditions.

1996 ◽  
Vol 40 (3) ◽  
pp. 621-626 ◽  
Author(s):  
J E Sjöström ◽  
J Fryklund ◽  
T Kühler ◽  
H Larsson
Keyword(s):  
Antibacterial Activity ◽  
Proton Pump Inhibitors ◽  
Proton Pump ◽  
Bactericidal Activity ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Antibacterial Activities ◽  
In Vitro Antibacterial Activity ◽  
H Pylori

Factors affecting the in vitro antibacterial activity of omeprazole were studied. Our data show that 3H-labeled omeprazole covalently bound to Helicobacter pylori and to other gram-negative and gram-positive bacteria. The compound was found to bind to a broad range of proteins in H. pylori, and at pH 5, binding was enhanced 15-fold compared with binding at pH 7. The bactericidal activity correlated to the degree of binding, and at pH 5, a pH at which omeprazole readily converts to the active sulfenamide form, beta-mercaptoethanol, a known scavenger of sulfenamide, and fetal calf serum, to which noncovalent protein binding of omeprazole is known to occur, reduced the level of binding and almost entirely abolished the bactericidal activity. At pH 7 the killing activities of omeprazole and structural analogs (e.g., proton pump inhibitors) were dependent on the time-dependent conversion (half-life) to the corresponding sulfenamide. The bactericidal activity exerted by the sulfenamide form at pH 5 was not specific for the genus Helicobacter. However, in brucella broth at pH 7 with 10% fetal calf serum, only Helicobacter spp. were susceptible to omeprazole, with MBCs in the range of 32 to 64 micrograms/ml, and MBCs for more stable proton pump inhibitors were higher. Wild-type H. pylori and its isogenic urease-deficient mutant were equally susceptible to omeprazole. Thus, the urease is not a lethal target for omeprazole action in H. pylori. In conclusion, the antibacterial activities of omeprazole and analogs are dependent on pH and the composition of the medium used. Thus, at a low pH in buffer, these compounds have a nonselective action, whereas in broth at neutral pH, the mechanism of action is selective for Helicobacter spp.

scholarly journals Delaying meiotic resumption during transportation of bovine cumulus–oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 740-750 ◽  
Author(s):  
Priscila Chediek Dall'Acqua ◽  
Beatriz Caetano da Silva Leão ◽  
Nathália Alves de Souza Rocha-Frigoni ◽  
Fernanda Patrícia Gottardi ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Bovine Serum Albumin ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
The Other ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Control Groups ◽  
Meiotic Inhibitors

SummaryThis study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 μM butyrolactone I (BL), 500 μM IBMX + 100 μM forskolin (mSPOM), 100 μM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 μM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P < 0.05) were obtained in the Control Lab FCS group (84.5 ± 5.3%) compared with the other groups (59.6 ± 3.4% to 70.9 ± 2.3%). Embryonic development was higher (P < 0.05) in the Control Lab FCS group (39.8 ± 4.7%) than in the Control Transp BSA (22.7 ± 3.4%) and MR (21.6 ± 2.3%) groups. However, they were similar (P > 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P < 0.05) in the Control Lab FCS group (85.2 ± 5.6) than in Control Lab BSA (53.6 ± 2.9), Control Transp BSA (55.5 ± 4.4), BL (58.2 ± 3.0), mSPOM (57.9 ± 4.9) and MR (59.2 ± 3.9), but all these treatments did not differ (P > 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Fetal Calf Serum ◽  
Formation Rate ◽  
Calf Serum ◽  
Basic Medium ◽  
Intracellular Camp ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
High Concentration

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.

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