Mccoy s 5a serum free medium

Manufactured by Merck Group

Sourced in United States

McCoy's 5A serum-free medium is a cell culture medium formulated to support the growth and maintenance of various cell lines in the absence of serum. It provides a standardized, chemically-defined environment for cell culture applications.

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McCoy’s 5A (83 citations) McCoy’s medium (25 citations) McCoy 5A medium (12 citations) McCoy 5A (10 citations) Serum-free medium (9 citations) McCoy medium (4 citations)

3 protocols using mccoy s 5a serum free medium

Colon Cancer Cell Line Culture

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The human colon carcinoma RKO, HCT116, FET and GEO cell lines were cultured in McCoy's 5 A serum-free medium (Sigma, St Louis, MO, USA) supplemented with 10 ng/ml epidermal growth factor, 20 μg/ml insulin and 4 μg/ml transferrin.^{59 (link)} HCT-116 p53^−/− cell line was cultured in McCoy's 5 A serum-free medium supplemented with 10% of fetal bovine serum (Life Technologies, Grand Island, NY, USA). Cells were maintained at 37°C in a humidified incubator with 5% CO₂. 5-FU and MK2206 were purchased from Sigma and Selleckchem (Houston, TX, USA), respectively. Anti-hsa-miR-587 miScript miRNA inhibitor was purchased from Qiagen Inc. (Valencia, CA, USA). Antibodies were purchased as indicated: anti-PPP2R1B (IHC), Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); anti-PPP2R1B (western blot), Abcam (Cambridge, MA, USA); anti-PARP, anti-cleaved PARP, anti-AKT, anti-pAKT(T308), anti-pAKT(S473), anti-GSK3β, anti-pGSK3β(S9) anti-XIAP, Cell Signaling Technology (Beverly, MA, USA); and anti-actin, Sigma.

Zhang Y., Talmon G, & Wang J. (2015). MicroRNA-587 antagonizes 5-FU-induced apoptosis and confers drug resistance by regulating PPP2R1B expression in colorectal cancer. Cell Death & Disease, 6(8), e1845-.

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Validation of Colon Cancer Cell Lines

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Human colon carcinoma cell lines (HCT116, RKO, FET, CBS, HCT116b and TENN) were originally established in Dr. Brattain’s lab in 1981 (27 (link)). All cell lines were validated by short-tandem repeat (STR) analyses at the University of Nebraska Medical Center Human DNA Identification Laboratory. STR profiles were cross-checked with the ATCC database. HCT116 and RKO cell lines with ≥ 80% match with the ATCC online STR database are considered valid (28 (link)). STR profiles of FET, CBS, HCT116b and TENN cell lines have never been reported before. Their STR profiles were confirmed in ATCC database to be of human origin and contain no mammalian interspecies contamination (Fig. S1A). There were 5–8 passages between thawing and use in the described experiments for all the cell lines.
Cells were maintained at 37°C in a humidified incubator with 5% CO2 and cultured in McCoy’s 5A serum-free medium (M4892, Sigma, St. Louis, MO, USA) supplemented with 10 ng/ml epidermal growth factor, 20 μg/ml insulin, and 4 μg/ml transferrin (29 (link)). When the cells were subjected to growth factor deprivation stress, they were cultured in the medium in the absence of growth factor or serum supplements.

Zhou X., Geng L., Wang D., Yi H., Talmon G, & Wang J. (2017). R-Spondin1/LGR5 Activates TGFβ Signaling and Suppresses Colon Cancer Metastasis. Cancer research, 77(23), 6589-6602.

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Colon Cancer Cell Line Culturing and Treatments

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The human colon cancer cell lines, FET, FET-DNRII, CBS, and CBS-RII, were described previously [32 (link)]. All cells were cultured in McCoy’s 5A serum-free medium (Sigma) supplemented with 10 ng/ml epidermal growth factor (EGF), 20 μg/ml insulin, and 4 μg/ml transferrin [44 (link)]. When cells were subjected to growth factor deprivation stress (GFDS), they were cultured in McCoy’s 5A serum-free medium in the absence of EGF, insulin and transferrin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. TGFβ and the TGFβ RI kinase inhibitor were obtained from R&D Systems and Calbiochem respectively. The following antibodies were used in this study: anti-IRS-1 (1:1000), Cell Signaling #2382, anti-p-IRS-1Y632 (1:600), Santa Cruz sc-17196, anti-p-IRS-1/2Y612 (1:600), Santa Cruz sc-17195-R, anti-Smad3 (1:1000), Cell Signaling #9523, anti-p-Smad3S423/425 (1:1000), Cell Signaling #9520, anti-p-Smad2S465/467 (1:1000), Cell Signaling #3101, anti-XIAP (1:1000), Cell Signaling #14334, anti-Cyclin D1 (1:1000), Cell Signaling #2978 and anti-Actin (1:1000), Sigma #A2066.

Bailey K.L., Agarwal E., Chowdhury S., Luo J., Brattain M.G., Black J.D, & Wang J. (2017). TGFβ/Smad3 regulates proliferation and apoptosis through IRS-1 inhibition in colon cancer cells. PLoS ONE, 12(4), e0176096.

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