All cell lines were cultured in media supplemented with 10% Heat inactivated FBS (ThermoFisher, 16140071) and 1% Pen/Strep (ThermoFisher, 15070063) at 37°C/90% RH/5% CO2, except for MDA-MB-468, which was cultured at 0% CO2. All cell lines are immortalized human cell lines, except for CT26, which is an immortalized mouse cell line. Sex, media, and RRID for each cell line: HAP1 [sex: male, media: IMDM (ThermoFisher, 31980030), RRID: CVCL_Y019]; HCT116 [sex: male, media: McCoy’s 5A (ThermoFisher, 16600108), RRID: CVCL_0291]; HT-29 [sex: female, media: McCoy’s 5A, RRID: CVCL_0320]; HCC1143 [sex: female, media: RPMI 1640 (ThermoFisher, A1049101), RRID: CVCL_1245]; A549 [sex: male, media: DMEM (ThermoFisher, 10569010), RRID: CVCL_0023]; HCC366 [sex: female, media: RPMI 1640, RRID: CVCL_2059]; HCC1954 [sex: female, media: RPMI 1640, RRID: CVCL_1259]; SKBR3 [sex: female, media: McCoy’s 5A, RRID: CVCL_0033]; MDA-MB-468 [sex: female, media: Leibovitz L-15 (ThermoFisher, 11415064), RRID: CVCL_0419]; NCI-H1650 [sex: male, media: RPMI 1640, RRID: CVCL_1483]; CT26 [sex: female, media: RPMI 1640, RRID: CVCL_7254]. Cell lines were tested for mycoplasma upon arrival at Pfizer and authenticated by STR analysis internally at Pfizer and/or by ATCC.
Mccoy s 5a
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom, Germany, Italy, Japan
McCoy's 5A is a cell culture medium formulated for the growth and maintenance of various cell types. It provides a balanced nutrient solution to support cellular functions and proliferation. The medium is designed to maintain the physiological pH and osmolarity required for optimal cell culture performance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (151 mentions)
Rpmi 1640, by Thermo Fisher Scientific (103 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (88 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (53 mentions)
Penicillin, by Thermo Fisher Scientific (52 mentions)
Streptomycin, by Thermo Fisher Scientific (52 mentions)
Hct116, by American Type Culture Collection (38 mentions)
Hct116, by Thermo Fisher Scientific (35 mentions)
Dmem f12, by Thermo Fisher Scientific (24 mentions)
Skov3, by American Type Culture Collection (20 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (19 mentions)
Skov3, by Thermo Fisher Scientific (16 mentions)
Sw480, by American Type Culture Collection (16 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (14 mentions)
L glutamine, by Thermo Fisher Scientific (13 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (12 mentions)
Skbr3, by American Type Culture Collection (11 mentions)
Dld 1, by American Type Culture Collection (11 mentions)
Sw480, by Thermo Fisher Scientific (11 mentions)
Hek293t, by Thermo Fisher Scientific (11 mentions)
347 protocols using mccoy s 5a
1
Cell Line Culture Conditions
2
Generating GSK5032-Resistant Cell Lines
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HCT116 parental and RKO parental cells were obtained fresh from ATCC (ATCC® CCL-247™, ATCC® CRL-2577™; Manassas, VA) and cultured in McCoy’s 5A (Life Technologies, 16600–082; Waltham MA) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, and RPMI (Gibco, 11875–093; Gaithersburg, MD) supplemented with MEM Non-Essential Amino Acids (Life Technologies, 11140–050; Waltham MA), sodium pyruvate (Life Technologies, 11360-070; Waltham MA), 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, respectively. Cells were treated with increasing doses of GSK5032 (GlaxoSmithKline; Collegeville, PA) starting with 200 nM and ultimately reaching 7400 nM over the course of 150 days. Fresh media and inhibitor were given to the cells every 3–4 days, passing the cells as needed. All cell lines tested negative for Mycoplasma contamination throughout the duration of the experiments. HCT116 and RKO GSK5032-resistant cell lines are available upon request. HCT116 DKO1 cells were kindly provided by Dr. Stephen B. Baylin and cultured in McCoy’s 5A (Life Technologies, 16600-082; Waltham MA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin.
3
Comparative analysis of sarcoma and lung cancer cell lines
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Sarcoma cell line RD, SW982 and corresponding knock-out cell lines BAXKO, BAKKO and BOKKO [26 (link)] were maintained in medium (DMEM; Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Germany) and 1% Penicillin/Streptomycin (Gibco, Life Technologies, Darmstadt, Germany). H1299/WT and H1299/TP53 were maintained in RPMI 1640 (RPMI 1640; Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% FCS and 1% Penicillin/Streptomycin. HCT116 and corresponding knock-out cell lines BAXKO, BAKKO and BAXKO/BAKKO were maintained in McCoy´s 5 A (McCoy´s 5 A; Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% FCS and 1% Penicillin/Streptomycin. Human STS cell line SW982 was authenticated by STR-profiling at the DSMZ. Cells were harvested after incubation in 0.05% trypsin/EDTA solution, centrifuged at 800 x g for 5 min and further processed for subsequent analysis.
4
Cell Culture Conditions for Various Cell Lines
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HEK-293T, G401, TTC1240, ESX, IMR-90, BJ Fibroblast, CRL7250, and NCIH-1437 cells were grown in DMEM (Gibco) supplemented with 10% FBS, 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco). ES-2 cells were grown in McCoy’s 5A (Gibco) supplemented with 10% FBS, 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco). EoL-1 and MOLM-13 were grown in RPMI (Gibco) supplemented with 10% FBS, 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco). RD were cultured in DMEM (Gibco) supplemented with 10% FBS. HCT116 were grown in McCoy’s 5A (Gibco) supplemented with 10% FBS. Calu-6 were grown in EMEM (ATCC 30–2003) supplemented with 10% FBS. SYO-1 was grown in DMEM without sodium pyruvate (Gibco) supplemented with 10% FBS, 1% GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco). The SYO-1 cell line was a gift from Akira Kawai (National Cancer Center Hospital, Japan. ASKA and HS-SY-II cell lines were obtained from RIKEN49 (link). The CRL7250 human fibroblast cell line was a kind gift from Drs. Berkeley Gryder and Javed Khan (National Cancer Institute, Bethesda, MD).
5
Cell Culture Protocols for Intestinal and Colorectal Cancer
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Human normal intestinal epithelial cells (NCHM460) were obtained from IMMOCELL in Xiamen, China, and human colon cancer cell lines (Caco2, HCT15, HCT116, HT29, Lovo, SW480, SW620) were obtained from icell in Shanghai, China. All these cells were cultured in media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C in a humidified atmosphere of 5% CO2. The culture media used were DMEM, MEM, 1640, McCOY’s 5A, McCOY’s 5A, F12K, L15 and L15, which were purchased from Gibco BRL in the USA.
6
Cell Culture Conditions for Various Cell Lines
7
Akt1/2 KO HCT116 Cell Line Transfection
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The Akt1/2 knockout HCT116 colon cancer cell line was given as a gift from Dr Bert Vogelstein (Johns Hopkins University; Ericson et al., 2010 (link)). Cells were cultured in McCoy’s 5A (Gibco) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco) at 37°C and 5% CO2. When the cell confluence reached around 70% in six-well plates, the medium was changed with McCoy’s 5A (Gibco) having 5% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco), and the cells were transfected with 1.5 μg of pcDNA3.1-Flag-HA-Akt plasmid or 1.5 μg (3.0 μg for Y18A mutant) of pcDNA3.1-Myr-HA-Akt plasmid complexed with 3 μL Lipofectamine 3000 (Invitrogen) and 3 μL P3000 reagent (Invitrogen) in Opti-MEM medium (Gibco) at 37°C and 5% CO2. Seventy-two hours after transfection, the cells were washed with cold PBS and lysed by adding 150 μL RIPA buffer (Cell Signaling Technology) containing ×1 complete protease inhibitor tablet (Thermo Fisher Scientific) and ×1 PhosStop tablet (Roche), and 1 mM PMSF for 10 min at 4°C. Thirty μg of total protein (BCA assay) was loaded on an SDS-PAGE gel. Membrane transfer and western blotting were carried out as described above with 1:1000 dilution for primary antibodies: anti-Akt, anti-pT308 Akt, anti-Foxo1, anti-Foxo3a, and anti-pT24 Foxo1/pT32 Foxo3a, anti-GAPDH (Cell Signaling Technology).
8
Follicle Culture Protocol: Optimizing Conditions
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All chemicals and media were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless mentioned otherwise.
The basal medium (BM) was McCoy's 5A supplemented with 10% FBS (GIBCO, Grand Island, NY, USA), 50 mg/mL streptomycin, 2 mmol/L glutamine, and 100 IU/mL penicillin. The follicle culture medium (FCM) was McCoy's 5A supplemented with 10% FBS, 50 mg/mL streptomycin, 2 mmol/L glutamine, 100 IU/mL penicillin, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 4 ng/mL FSH, 3 mg/ mL BSA(GIBCO, Grand Island, NY, USA), 50 μg/ mL ascorbic acid, and 2 mmol/L hypoxanthine.
The pH of the medium was adjusted between 7.2 and 7.4 before filtration, following which the medium was sealed at a temperature of 4°C for preservation.
The basal medium (BM) was McCoy's 5A supplemented with 10% FBS (GIBCO, Grand Island, NY, USA), 50 mg/mL streptomycin, 2 mmol/L glutamine, and 100 IU/mL penicillin. The follicle culture medium (FCM) was McCoy's 5A supplemented with 10% FBS, 50 mg/mL streptomycin, 2 mmol/L glutamine, 100 IU/mL penicillin, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 4 ng/mL FSH, 3 mg/ mL BSA(GIBCO, Grand Island, NY, USA), 50 μg/ mL ascorbic acid, and 2 mmol/L hypoxanthine.
The pH of the medium was adjusted between 7.2 and 7.4 before filtration, following which the medium was sealed at a temperature of 4°C for preservation.
9
Cell Line Cultivation and Maintenance
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SKOV3, OVCAR3, MCF7, MDA231, T47D were obtained from the ATCC (Manassa, Virginia, USA). SKOV3 (ATCC-HBT-77) was grown in McCOY's 5a (Life technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS, Wisent technologies, St-Bruno, Quebec
Canada). MCF7 (ATCC-HTB-22) and MDA231 (ATCC-HTB-26) cells were grown in DMEM/F12 (Life technologies), supplemented with 10% FBS. T47D (ATCC-HTB-133) and OVCAR3 (HTB-161) cells were grown in RPMI-1640 (Life technologies), supplemented with 10% and 20% FBS, respectively. Culture method was followed as described by ATCC for each cell line. Kuramochi (JCRB No. JCRB0098) and OVSAHO (JCRB No. JCRB1046) cells were obtained from JCRB (Japanese Collection of Research Bioresources) Cell Bank and Sekisui Xenotech LLC (Cambridge, Kansas City, USA). Cells were grown in RPMI-1640 (Life technologies) supplemented with 10% FBS.
Canada). MCF7 (ATCC-HTB-22) and MDA231 (ATCC-HTB-26) cells were grown in DMEM/F12 (Life technologies), supplemented with 10% FBS. T47D (ATCC-HTB-133) and OVCAR3 (HTB-161) cells were grown in RPMI-1640 (Life technologies), supplemented with 10% and 20% FBS, respectively. Culture method was followed as described by ATCC for each cell line. Kuramochi (JCRB No. JCRB0098) and OVSAHO (JCRB No. JCRB1046) cells were obtained from JCRB (Japanese Collection of Research Bioresources) Cell Bank and Sekisui Xenotech LLC (Cambridge, Kansas City, USA). Cells were grown in RPMI-1640 (Life technologies) supplemented with 10% FBS.
10
Culturing Osteoblast Progenitor Cells
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MC3T3-E1 mouse calvarial preosteoblasts were obtained from the Cell Center of the Chinese Academy of Science (Shanghai, China). Human osteosarcoma cell lines U-2OS, murine monocytic cell lines RAW 264.7, and murine mesenchymal C3H10T1/2 cells were purchased from the Cell Center of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). MC3T3-E1, U-2OS, RAW264.7, and C3H10T1/2 cells were cultured in α-MEM, McCoy’s 5A, DMEM, and MEM, respectively, with 10% FBS (Life Technologies, Carlsbad, CA, USA). Osteogenic differentiation of MC3T3-E1 cells was induced using an osteogenic induction medium containing α-MEM supplemented with 10% FBS, 50 mg/ml ascorbic acid, and 10 mM β-glycerophosphoric acid. All cells were cultured at 37°C in a 5% CO2 atmosphere.
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