McCoy’s medium

Grow the HT-29 cell line in T75 flasks in McCoy s 5A medium (10% FBS). When cells are confluent, harvest HT-29 cells by adding 2.0 ml of TrypLE Express. After the cells detach from the flasks, resuspend the cells in McCoy s medium (10% FBS) to inactivate the TrypLE Express. Count the cell concentration as described below. Prepare two small T25 flasks with McCoy s medium (10% FBS) and seed 1 x 106 cells per flask. Keep the HT-29 cells in culture as they will be needed again later. 

Replace the medium every 3 days with fresh McCoy s medium (10% FBS) supplemented with 800 pg/ml of geneticin for the next 2 weeks. 

Thus McCoy s medium 5A (McCoy et al., 1959) has been used as a standard medium for cloning cells. It is based on BME amino acids and the vitamins from medium 199 (Appendix 1 Table 18). This was modified further to form RPMI (Rosswell Park Memorial Institute)-1629 (Appendix 1 Table 17) for long-term culture of leukaemic myoblasts (Armstrong, 1966). 

Exponentially growing cells are used and it is important to maintain constant pH and temperature throughout the selection procedure. Klevecz et al. (1974, 1975) have used CHO cells growing in McCoy s medium with 20% foetal bovine serum and Hepes buffer. They maintain the cells throughout in this medium at 37°C and, of the cells they select, 98-99% are in mitosis. The viability of these selected cells approaches 100% and on reseeding half the cells attach within 1 h of selection and maximum attachment is found by 4 h (Klevecz, 1975). However, they select a very small proportion of the original cells. 

Inoculate cells into bottles 24 h prior to initial selection. 5 X 107 Don-C Chinese hamster fibroblast cells may be inoculated into a roller bottle in McCoy s medium (see 11.2.2). 

In hydrophobic teflon bags, the test compound and blood monocytes isolated from pooled buffycoats at a density of 2 x 10 -cells/mL McCoy s medium supplemented with 20% fetal calf serum are cultured for 2 days. The monocytes are washed with... 

Plant 1 ml suspended cells in Leighton tubes and add 1 ml McCoy s medium or plant 5 ml suspended cells in 30-ml Falcon flasks. 

Feed once a week with McCoy s medium, until good growth is... 

Figure 4.98 was obtained from a system where a solution consisting of McCoy s medium with 10% AB serum was further supplemented with a final concentration of 10% of DMSO. It can be seen that the exothermic peak (-56.2 °C and 12.3 Jg ) appearing in Figure 4.97 disappeared. 

SK-BR-3 This is an adherent breast carcinoma cell line. This cell line is cultured in McCoy s 5A medium with 2mM glutamine and 10% Fetal Bovine Serum (FBS) at 37°C in a 5% C02 in air atmosphere. 

HCT-116 human colon carcinoma (ATCC, Bethesda MD) cells were grown in McCoy s 5A) and were routinely subcultured twice weekly. Antiproliferative assay was performed by chemoluminescence assay based on quantification of ATP. Cells in their exponential phase of growth were treated at different times (lh or 24h) with different concentrations of edotecarin or SN-38. For post-treatment recovery studies, cells were washed with PBS and left in drug-free culture medium. Then, cell medium was collected to avoid any cell loss. Cells in monolayer were washed, detached with trypsin, and collected in the medium. Cells were counted in a Multisizer 3 Coulter Counter to measure the drug s effects on growth inhibition. Samples were fixed either... 

Cell culture medium McCoy s 5A Medium with L-glutamine, 10% FCS. 

Aspirate the supernatant and resuspend the cell pellet in freezing medium (McCoy s 5A Medium with L-glutamine, 10% PCS, 5% DMSO) to a concentration of 1 x 10 cells/ml. 

After 30 h, add 5 pi ofSST-14 from a 9x stock solution in assay medium (McCoy s 5A, 0.2% PCS, no selection antibiotics). 

RPMI, modified McCoy 5 A medium for cell culture HEPES, 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid IMDM, Iscov s modified Dulbecco s medium PCS, fetal calf serum... 

Aspirate HT-29LP medium (McCoy s 5A, supplemented with 1% glutaMAX, 10% FBS, and 0.8 mg/ml geneticin), from adherent HT-29LP cells growing in a polystyrene flask or dish and add 2 ml TrypLE Express. Move the solution around the plate and quickly aspirate. 

Add medium (McCoy s 5A, supplemented with 1% Gluta-Max and 10% FBS without geneticin) to bring HT-29LP cells to the final desired concentration. For the utilized dose of 1 x 106 cells/mouse, the cells should be brought to 20 x 106 cells/ml. Put the cells on ice to bring to approximately 4°C. 

Subculture Don-C cells every 24 h in McCoy s 5a medium (Appendix 1) containing 20% foetal calf serum and 0.08% lactalbumin hydrolysate, seeding cells at 1.2 X 105/ml(3 X 104 cells/cm2). 

McCoy s 5a medium (RPMI 1629) (originally formulated by McCoy et al., 1985 and modified by Hsu and Kellogg, 1960 and Iwakata and Grace, 1964). 

Saos-2 cell culture growth medium. 420 mL McCoy s 5 A (ATCC) supplemented with 1.5 mM glutamine, 1.1 g sodium bicarbonate (final concentration 2.2 g/L), 5 mL penicillin and streptomycin stock solution (ree Subheading 2.1) (100 times diluted to have final 100 U/mL penicillin and 0.1 mg/mL streptomycin) and 75 mL fetal bovine serum from PAA see Subheading 2.1), final concentration of bovine serum is 15 % v v. 

Proliferating cultures of Chinese Hamster Ovary cells were exposed for 24 h to the compounds shown in the table. The cells were allowed to undergo twelve divisions in complete growth medium and then about 7 X 10 cells attached to the monolayer were placed in selection medium (McCoy s 5a medium supplemented with 10% fetal bovine serum, 8 yg/ml 8-azaguanine (8-AG) and 6 ug/ml 6-thioguanine (6-TG). Cultures were incubated in selection medium for about three (3) weeks. The selection medium was replenished with fresh medium about two (2) times each week. At the end of the selection period the cultures were washed two (2) times with normal saline, fixed with 95% ethanol and stained with an ethanol-crystal violet solution (0.5% w/v crystal violet in 95% ethanol). A colony was defined as a cluster of 50 or more cells. 

Flood gently with 5 ml medium (McCoy s with 30% fetal calf semm, 100 U/ml penicillin, and 100 / g/ml streptomycin). If any pieces float, remove and reimplant as above in fresh bottle. 

Resuspend the cell button with 5 to 7 ml McCoy s 5A medium (with 30% fetal calf serum and L-glutamine). 

Chinese hamster ovary (CHO) cells were maintained as monolayers in McCoy s 5A medium supplemented with 10% fetal calf serum and antibiotics. Normal human lymphoid cells, WIL-2 were maintained as suspension cultures in RPMI medium containing fetal calf serum and antibiotics. 

Fig. 1. NAD content of CF-3 cells incubated in Ca + depleted medium O— O), Ca + supplemented medium ( - ), and Ca + depleted medium supplemented with normal levels of Ca + (1.8 mM) after 2 hr of incubation (x—x). Arrow indicates time of Ca + addition. Values are Mean SEM for three experiments. Ca + depleted medium was McCoy s 5a prepared without nicotinamide, nicotinic acid, and CaCl2. It was supplemented with 10% FBS treated as described (14). NAD determinations were performed as described (14). Reprinted with permission from ref. 14.

McCoy s 5A medium (AMEM) alpha modification of Eagle s medium (see Hay et al., 1992, for formulas). The ... 

Figure 4.98 DSC curves of a cell-frcc system (10% AB serum-10% DMSO-80% McCoy s 5A medium. Sample mass, 3.9 mg cooling rate, 5 C min ...

CMRI 1066 McCoys 5A M 199 RPMI 1629 William s medium E Waymouth s medium... 

HeLa cells were cultured in a CO2 incubator by controlling the CO2 concentration at 5% at a constant temperature of 35 C. The culture medium was McCOY s 5A mixed with 10% PBS. Cultured HeLa cells were treated with trypsin, washed, and collected by centrifugation. 

PBS. After 10 min. the enzyme was rinsed out with McCoy s 5 a medium (GIBCO) and the segment was filled with complete medium (McCoy s 5a medium, supplemented with 10% fetal calf serum, 15 mM Hepes buffer, and O.lOmg/ml penicillin, O.lOmg/ml streptomycin, 0.20mg/ml neomycin, and 5.0 mcg/ml Fungizone), The calves were allowed to recover after the cells were removed. 

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