Breast cancer cell lines MDA-MB-231, –436, –468, MCF7, and non-tumorigenic breast epithelial cells, MCF10A, were purchased from the American Type Culture Collection and grown under recommended conditions. Patient-derived cancer cells were isolated in our lab (T4) (Gonzalez et al., 2014 (link)), Dr. Merajver lab (VARI-068) (Gilani et al., 2016 (link)) and provided by Dr. Wicha (GUM36). Patient-derived LN and Lv-MSC were isolated and characterized in our lab (Gonzalez et al., 2017 (link)). Human adipose MSCs, primary fibroblasts, and brain and dermal microvascular endothelial cells were purchased from ScienCell Research Laboratories (#7510, 7630, 1000, and 2020, respectively), and maintained following the provider’s instructions. Human immortalized fibroblasts were provided by Dr. Luker (Ray et al., 2015 (link)). Cells were delivered frozen after being isolated from normal human tissue and being cryopreserved at first passage. Cell lines were authenticated using STR profiling, and were tested for mycoplasma infection using Sigma LookOut Mycoplasma PCR Detection Kit (Cat MP0035).
Sigma lookout mycoplasma pcr detection kit
Manufactured by Merck Group
The Sigma LookOut Mycoplasma PCR Detection Kit is a laboratory equipment product designed for the detection of mycoplasma contamination in cell cultures. The kit utilizes polymerase chain reaction (PCR) technology to identify the presence of mycoplasma DNA. It provides a reliable and efficient method for monitoring cell culture samples for mycoplasma contamination.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (3 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Mda mb 468, by American Type Culture Collection (2 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Mycoplasma removal agent, by MP Biomedicals (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Mda mb 231, by National Centre for Cell Science (1 mentions)
Mcf 7, by National Centre for Cell Science (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Mda mb 453, by National Centre for Cell Science (1 mentions)
Mission shrna, by Merck Group (1 mentions)
Mda mb 436, by American Type Culture Collection (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Snai2 slug, by Cell Signaling Technology (1 mentions)
Puromycin, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
6 protocols using sigma lookout mycoplasma pcr detection kit
1
Comprehensive Breast Cancer Cell Characterization
2
IMCD3 cell culture protocol
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IMCD3 cells were grown in DMEM-F12 medium with 10% fetal bovine serum and 1% penicillin-streptomycin-kanamycin antibiotic cocktail at normoxic conditions. Cells were passaged every 2–3 days at a dilution of 1:10–1:20. Cells were tested for mycoplasma with Sigma LookOut Mycoplasma PCR Detection Kit (Cat# MP0035) as directed by the manufacturer, and incidences of mycoplasma contamination were treated with Mycoplasma Removal Agent (MP Biomedicals, #093050044). Following decontamination, experiments that were potentially affected by mycoplasma contamination were repeated at least three times to determine any difference in results, and no significant differences were observed.
3
Cell Line Characterization and Maintenance Protocol
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MDA-MB-231 (BRAF, c.1391G>T, p.G464V; KRAS, c.38G>A p.G13D), MCF-7 (PIK3CA, c.1633G>A, p.E545K), MDA-MB-361 (PIK3CA, c.1633G>A p.E545K) and MDA-MB-453 (BRAF, KRAS and PIK3CA wild type) were purchased from the National Centre for Cell Science (NCCS, Pune, India). The cell lines were routinely cultured in 75-mL tissue culture flasks (Nunc Thermofisher Scientific) in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (all from Sigma, USA) at 37°C and 5% CO2, according to the protocol provided by NCCS. All cell lines were tested for Mycoplasma detection by the Sigma LookOut Mycoplasma PCR Detection Kit (MP0035).
4
Hybrid Cell Formation Between Breast Cancer and Stromal Cells
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BCC lines MDA-MB-231, MDA-MB–436, MCF-7, and MDA-MB-468 were purchased from the American Type Culture Collection and grown under recommended conditions. Patient-derived cancer (T4) as well as MSCs isolated from fresh human breast cancer metastasis to a supraclavicular lymph node (LN-MSCs) and to the liver (Lv-MSCs) were isolated and characterized in our lab (13 (link)). CCN6-KO cells derived from MMTV-Cre;Ccn6fl/fl mammary breast carcinomas developed and validated in our lab (15 (link)) were cultured with DsRed-MSCs for 7 days. Cell lines were authenticated using STR profiling and were tested for mycoplasma infection using Sigma LookOut Mycoplasma PCR Detection Kit (catalog MP0035). BCCs and MSCs were labeled with GFP and DsRed, respectively. In another set of experiments, BCCs were labeled with DsRed. To generate hybrid cells, 2 × 105 DsRed-MSCs and 1 × 105 GFP -BCCs were cultured in complete medium (50% of BCC medium and 50% of MSC medium) for 72 hours or 7 days as indicated.
Human WNT5A knockdown was achieved using shRNA (MISSION shRNA, Sigma-Aldrich; Broad Institute constructs TRCN0000288987 and TRCN0000296083 for WNT5A) as previously reported (41 (link)). Background control was Lenti-PuroEMPTY-VSVG. Cells were selected with 1 μg/mL puromycin over time to eliminate uninfected cells. WNT5A protein expression in stable clones were evaluated by immunoblotting.
Human WNT5A knockdown was achieved using shRNA (MISSION shRNA, Sigma-Aldrich; Broad Institute constructs TRCN0000288987 and TRCN0000296083 for WNT5A) as previously reported (41 (link)). Background control was Lenti-PuroEMPTY-VSVG. Cells were selected with 1 μg/mL puromycin over time to eliminate uninfected cells. WNT5A protein expression in stable clones were evaluated by immunoblotting.
5
Cell Line Maintenance and Mycoplasma Testing
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The DU145, PC3, LNCaP, 293FT, and phoenix cells were purchased from ATCC (Manassas, VA). The DU145, PC3, and LNCaP cells were maintained in RPMI-1640/10% FBS/1% pen-strep media. The 293FT and phoenix-amphotropic cells were maintained in DMEM/10% FBS/1% pen-strep media. All cells were maintained in the exponential phase of growth at 37°C in a humidified 5% CO2 atmosphere. Tet-free FBS was used to maintain the Tet-ON HK2sh and Tet-ON control (shScr) cells in the absence of doxycycline, and doxycycline induction was at 900 ng/ml for the inducible DU145, PC3, and LNCaP HK2 knockdown cell lines.
All cells were confirmed to be mycoplasma negative, using the Sigma LookOut Mycoplasma PCR Detection Kit (Sigma, St. Louis, MO).
All cells were confirmed to be mycoplasma negative, using the Sigma LookOut Mycoplasma PCR Detection Kit (Sigma, St. Louis, MO).
6
Investigating EMT Regulators in Breast Cancer
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