Tissue culture media fetal bovine serum

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

Seek T75 plastic tissue culture flasks with a minimum of 2.5 x 106 cells in 120ml of Eagle s medium containing 20mM L-glutamine 0.88g l-1 sodium bicarbonate 20 mM HEPES 50 pg ml-1 streptomycin sulphate 50IUml 1 benzyl-penicillin and 7.5% fetal bovine serum. The flasks are incubated for 18-24 h at 37°C in a C02 incubator to establish monolayer cultures. 

Remove about 0.5 mL of the top most layer in the tube (the buffy coat of the plasma), and add it to a tissue-culture flask containing 5 mL of Ham s F-10 medium supplemented with 10% fetal bovine serum and 400 pL of phytohemagglutinin. Incubate for 72 h at 37°C in a carbon dioxide incubator (see Note 4). 

Media basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use. 

Procedure. An aliquot of the neutralized diazotized [ I]ISA is added to a washed, buffered suspension of cells (pH 7.5) at 5°. The reaction goes to completion within 15-30 min depending on the cells being labeled. The reaction is stopped by washing the cell suspension 3 or 4 times with a lOx volume of 0.15 M NaCl containing % bovine serum albumin or serum-containing tissue culture medium (e.g., RPMI 1640 with 10% fetal calf serum). 

A variety of in vitro toxicity tests have been developed to model the effects of toxins on living cells or tissues. In these tests, a carrier medium (such as fetal bovine serum) containing given concentrations, or doses, of a particular toxin are added to cell cultures (cell lines). Various indicators of toxicity, cell morphology transformation, or cell prohferation are then measured after specified periods of time. The cell types used in a particular study can be chosen to approximate the types of cells that would be affected during acmal exposure, such as respiratory cells or tissues. Toxicity indicators include, for example, measures of the percent of viable cells remaining at the end of the test (compared to a control line with no added toxin), and the concentrations various cytokines or other cytoplasmic enzymes induced from the cells by the toxin. Uncertainties with the in vitro toxicity tests include how comparable their results are to those of in vivo toxicity tests, and how well they reproduce actual physiological conditions and processes in the human body (Johnson and Mossman, 2001). 

Cell Culture. BSC-1 cells were grown in minimal essential medium (MEM) with Earle salts supplemented with 10% fetal bovine serum (FBS) and 1.1 g/1 sodium bicarbonate. Cells were passaged according to conventional procedures by using 0.05% trypsin plus 0.02 wt% ethylenedi-aminetetraacetic acid (EDTA) in a HEPES-buffered balanced salt solution. Tissue culture flasks were incubated at 37°C in a humidified 3% CO2 - 97% air atmosphere. Total cell counts were made using a Coulter counter equipped with a 100-pm orifice and microscopic cell count. 

For adherent cell cultures, the following protocol for HeLa cells may serve as an example. Cervical adenocarcinoma (HeLa) cells (purchased from the American Tissue and Cell Culture Corp. ATCC Manhasset, VA, USA), cell line CCL-2, were seeded in 75 cm sterile plastic cell culture flasks (Fisher Scientific) at a concentration of approximately 2 X 10 cells cm l Typically, the growth medium consisted of 20ml Dulbecco s Modified Eagle s Medium (DMEM ATCC), supplemented with 10% fetal bovine serum (FBS ATCC). To prevent bacterial contamination, 2.5ggmT of amphotericin B (ATCC) and lOOIUmT penicillin/streptomycin (ATCC) was added to the medium. The cells were incubated at 37 °C in an atmosphere containing 5% COj. 

Adherent cells were maintained in DMEM/F-12 culture medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin-100 /.tg/ml streptomycin at 37 °C and under 5% CO2. In our experiments, VNOf90 cells derived from rat vomeronasal organs were used. Two days before use, cells which were in a confluent state with a density of about 10 /cm were released from wells using 0.25%i trypsin-1 mM EDTA. A drop of cell solution (about 10 of the cells) was plated on the center of 35 mm tissue culture dishes and incubated for about an hour. After 1 h incubation, cells were weakly attached on dishes and the dish was then filled with culture medium. The cells were only located at the center of the dish when we performed mRNA extraction with the AFM. This procedure is quite important to decrease the probability of mRNA contamination, because dead or floating cells are one of the causes of contamination. Two hours before use, the cells were rinsed with culture medium without FBS two or three times, and the dish was filled with FBS-free culture medium. 

Tissue-culture media endotoxin-free Dulbecco s modified Eagles medium (DMEM), minimal essential medium-alpha (MEMa) or Hepes-buffered RPMI-1640 supplemented with 2 mM r-glutamine, and heat-inactivated fetal bovine serum (FBS, to 10% final concentration) (see Note 1). Store at 4 °C. 

Human embryonic palatal mesenchymal cells (HEPM, ATCC CRL-1486) were seeded onto the coated surfaces in multiple 24-well tissue culture plates (50,000 cells/well). The cultures were maintained in Dulbecco s modified eagles medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), L-glutamine (2 pmol/ml), penicillin G (100 U/ml), streptomycin sulfate (100 pg/ml) and amphotericin B (0.25 pg/ml). Replicate cultures were incubated at 37 C for 6, 24 and 72 hours and then harvested for analysis. 

To study the effect of CNT and fiber size on cell prohferation, silkworm silk natural micrometer sized fibers, silkworm silk nanofibers with and without CNT and spider silk nanofibers with and without CNT were used. Human chondrosarcoma cells (ATCC HTB94) were maintained in culture using DMEM (Dulbecco s modified Eagle s medium Mediatech) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 1% L-glutamine. For cell seeding on scaffolds, cells from the tissue culture flasks were trypsinized for 5 minutes at 37 °C, neutralized with DMEM, centrifuged for 5 minutes at 1200 rpm and resuspended in DMEM. A sum of 1000000 cells was seeded per scaffold over 48 hours on a shaker at 37 C. The culture medium was supplemented with ascorbic acid (40 pg/ml) on the first day. The cell-scaffold constructs were maintained in the culture environment and... 

EAC-cells were kindly supplied from the National Cancer Institute, Cairo University. The cells were grown on the basal nutritional medium RPMI-1640 containing 1% gentamicin, 0.4% L-glutamine and 10% fetal bovine serum. Growth was continued till the cell density reached 1X10 cells/cm in sterile tissue culture flasks (Falcon). 

Endothelial cells are maintained in a basal culture medium, such as DMEM or a proprietary medium supplied with the cells, which is supplemented with hydrocortisone (lug/mL). epidermal growth factor (EGF 100 ug/mL) bovine fibroblast growth factor (FGF 1 ng/mL), antibiotics (gentamicin and amphotericin, at 50 ug/mL) and fetal calf serum (2-10%). Alternatively, bovine brain extract (3 ug/mL) can be used instead of EGF and FGF. The cells are cultured in either standard tissue culture flasks directly on plastic or on flasks coated with collagen. The cells are grown to confluence and subcultured and reseeded in a ratio of 1 3. For all experiments primary endothelial cells should be used between passages 3 and 12. 

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