U2OS cells (American Type Culture Collection, ATCC) were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum. MCF-10A cells (ATCC) were cultured in mammary epithelial growth medium containing insulin, hydrocortisone, epidermal growth factor, and bovine pituitary extract (Clonetics). EVSAT cells (Creative Bioarray, NY, USA) were cultured in MEM containing 10% fetal bovine serum. MDA-MB-436 cells (ATCC) were maintained in DMEM medium supplemented with 10% fetal bovine serum. PC3, DU145, ACHN, 786-0, H226, H522, OVCAR-3, OVCAR-8, and MCF7 cells were all obtained from ATCC and maintained according to ATCC instructions. BRCA1 (D-9) monoclonal and TTK polyclonal antibodies were purchased from Santa Cruz (SC-6954, 1:1000) and Cell Signaling (#3255, 1:1000), respectively. ZNF668 antibodies were generated as previously described41 (link). Uncropped scans of the most important western blots are listed as supplementary figures in Supplementary Figure 13 . PI3K inhibitor LY-294002 and mTOR inhibitor rapamycin were purchased from Sigma. PARP inhibitors olaparib and rucaparib, HDAC inhibitor vorinostat and Hsp90 inhibitor AUY922 were from Selleckchem. TTK inhibitor AZ3146 was purchased from R&D Systems.
Bovine pituitary extract
Manufactured by Cambrex
Bovine pituitary extract is a biological material derived from the pituitary glands of bovine (cattle) sources. The pituitary gland is responsible for the production of various hormones in the body. The extract contains a complex mixture of these hormones and other biologically active substances.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Cambrex (3 mentions)
Vorinostat, by Selleck Chemicals (2 mentions)
Rucaparib, by Selleck Chemicals (2 mentions)
Mcf10a cells, by American Type Culture Collection (2 mentions)
Ovcar8, by American Type Culture Collection (2 mentions)
Mda mb 436 cells, by American Type Culture Collection (2 mentions)
Brca1 d 9 monoclonal and ttk polyclonal antibodies, by Santa Cruz Biotechnology (2 mentions)
Auy922, by Selleck Chemicals (2 mentions)
Olaparib, by Selleck Chemicals (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Mtor inhibitor rapamycin, by Merck Group (2 mentions)
Du145, by American Type Culture Collection (2 mentions)
Ly294002, by Merck Group (2 mentions)
U2os cell, by American Type Culture Collection (2 mentions)
Ursodeoxycholic acid, by Merck Group (1 mentions)
Hydrocortisone, by Cambrex (1 mentions)
3 protocols using bovine pituitary extract
1
Cell Culture Protocols for Cancer Research
2
Cell Culture Protocols for Cancer Research
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U2OS cells (American Type Culture Collection, ATCC) were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum. MCF-10A cells (ATCC) were cultured in mammary epithelial growth medium containing insulin, hydrocortisone, epidermal growth factor, and bovine pituitary extract (Clonetics). EVSAT cells (Creative Bioarray, NY, USA) were cultured in MEM containing 10% fetal bovine serum. MDA-MB-436 cells (ATCC) were maintained in DMEM medium supplemented with 10% fetal bovine serum. PC3, DU145, ACHN, 786-0, H226, H522, OVCAR-3, OVCAR-8, and MCF7 cells were all obtained from ATCC and maintained according to ATCC instructions. BRCA1 (D-9) monoclonal and TTK polyclonal antibodies were purchased from Santa Cruz (SC-6954, 1:1000) and Cell Signaling (#3255, 1:1000), respectively. ZNF668 antibodies were generated as previously described41 (link). Uncropped scans of the most important western blots are listed as supplementary figures in Supplementary Figure 13 . PI3K inhibitor LY-294002 and mTOR inhibitor rapamycin were purchased from Sigma. PARP inhibitors olaparib and rucaparib, HDAC inhibitor vorinostat and Hsp90 inhibitor AUY922 were from Selleckchem. TTK inhibitor AZ3146 was purchased from R&D Systems.
3
Culturing of Normal Human Keratinocytes
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Normal human epidermal keratinocytes (NHEKs) were purchased from Clonetics (CC-2501) and Promocell. We used cells from 3 different donors of different race and age (referred as 4F0315, 2F1958, and K1MC). Cells were obtained anonymously and informed consent of each skin donor was obtained by the supplier. Cells were grown at 37°C in an atmosphere of 5% CO2 in a KGM-2 BulletKit medium consisting of modified MCBD153 with 0,15 mmol/L calcium, supplemented with bovine pituitary extract, epidermal growth factor, insulin, hydrocortisone, transferrin, and epinephrin (Clonetics). Such a serum-free low-calcium medium was shown to minimize keratinocyte terminal differentiation [29 (link)]. In all experiments, cells were seeded at 3500 cells/cm2 and always splitted at 70% confluence. The number of population doublings (PD) was calculated at each passage by means of the following equation: PD = log (number of collected cells/number of plated cells)/log2. Ursodeoxycholic acid and 3-(4-(Trifluoromethyl) phenylamino) benzoic acid were obtained from Sigma and Calbiochem, and diluted in ethanol and DMSO respectively.
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