Tissue culture media RPMI 1640 medium

Tissue culture medium (RPMI-1640) Dutch modified solution without glutamine (Gibco Life Sciences) with 2% PSF and 1% FCS added. 

M6 tissue culture medium RPMI 1640 (Sigma) L15 medium (Sigma), 50 50. 

Complete medium Tissue-culture basic salt medium (e.g., RPMI, EMEM, DMEM), with serum (e.g., 10% fetal calf v/v final concentration). 

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). 

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. 

Examine the cells under the microscope to ensure that all the fibroblasts are attached to the tissue culture dishes (Fig. la). Wash the layer of fibroblasts twice with 5 ml of pre-warmed PBS. Seed 2x10 CLL cells (ratio 10 1 10 CLL cells per NIH3T3 cell Fig. lb) on WT and CD154 N1H3T3 in 5 ml complete RPMI medium supplemented with 10 ng/ml of lL-4 (only in CD154 NIH3T3-coated tissue culture dishes) and incubate for 24 h in a humidified atmosphere of 5% CO at 37°C (see Notes 7 and 8). 

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. 

Quickly thaw a frozen aliquot of NS-O cells either in a 37°C water bath or between the palms of the hands. Once the pellet has melted, add 1 mL of 37°C RPMI1640 medium supplemented with 10% FBS and draw up into a Pasteur pipet. Transfer the cells to a 225-cm T flask and add 50 mL of RPMI 1640 medium containing 5% FBS and place in a tissue culture incubator 37°C/5% C02. 

CCRF-CEM T-lymphoblastoid cells (ATCC CCL 119) were cultivated in RPMI 1640 medium supplemented with L-glutamine (0.3 g/L) containing 10% bovine serum using 24-well tissue culture plates. The cells were seeded at 10s mL 1 and after a 24-h incubation period (C02 atmosphere, 37 °C) tested compounds were added at five different concentrations. The endpoint of the cell growth was 72 h following the drug addition. An appropriate aliquot from every dish was then counted (cell counter Serono 150+). The inhibitory potency of the compounds tested was expressed as IC50 values. 

Grow P3HR-1 cells in tissue culture flasks (max. 100 mL for 75-cm2 T-flask and 200 mL for 162-cm2 T-flask) in RPMI-1640 medium containing 10% FCS supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, and 100 pg/mL of streptomycin. 

Count the cells using a plastic disposable counting chamber and seed cells at 2 x 106 cells/mL in warmed RPMI growth medium in a 75-cm2 tissue culture flask. 

Plate the DCs in six-well tissue culture plates at 2x 10 cells per well in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (see Note 8). 

Human transferrin receptor-positive target cells are cultured at 37°C with 5% C02 in tissue culture flasks containing RPMI-1640 medium with 10% fetal calf serum, L-glutamine, and penicillin-streptomycin (GIBCO, Grand Island, NY). Adherent cells are released from the culture flask using 0.05% trypsin, and 0.02% EDTA for 10 min at 37°C, and then medium plus serum is added to inactivate the trypsin. 

RPMI 1640 media, Ham s F12K media, and Eagle s Minimal Essential Medium, MEME, respectively. All cell lines were grown in a tissue culture incubator supplied with 5% C02 and maintained at 37°C. 

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. 

Note the monocytes will differentiate into adherent macrophages by day 7 of incubation. To harvest cells, discard the culture medium and wash once with 25 mL of cold PBS without Ca++ or Mg++. Add 20 mL of fresh cold PBS to the flask and incubate on ice until cells begin to round-up and detach (usually 15-20 min) (see Note 14). Gently scrape cells into the PBS with a cell scraper and transfer the cell suspension to a 50-mL conical centrifuge tube. Pellet the cells by centrifugation at 500 x g for 5 min. Resuspend cells in 5-10 mL of RPMI plus 10% FBS and 50 ng/mL M-CSF. Count cells, add medium to adjust to the desired cell concentration, and re-plate in a tissue culture plate (see Note 15). Incubate at 37 °C in 5% CO2 for at least 6 h to allow macrophages to adhere. 

Ascorbate. The stock solution of 0.06 M L-ascorbate was made by dissolving L-ascorbic acid (tissue-culture grade from Sigma) in RPMI 1640 medium and was stored at -20°C. 

HeLa cells are grown on 10 cm tissue culture dishes for 2 days to 90% confluence in RPMI medium containing 10 mM Hepes, pH 7.3, and 10% FBS. After rinsing cells once with warm complete MEM medium, 10 ml of warm complete MEM medium is added and the dishes are incubated for 1 h at 37° in a CO2 incubator. 

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). 

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