The MM cell lines, MM1S, OPM2, OPM1, H929, OCIMY5, RPMI, U266, KMS1, HSB2, McCAR and ANBL6, and a breast cancer cell line MDA-MB-231 were obtained from ATCC (Manassas, VA). The T2 cell line, a human B and T cell hybrid expressing HLA-A2 molecules, was provided by Dr. J. Molldrem (University of Texas M. D. Anderson Cancer Center, Houston, TX). The cell lines were cultured in DMEM (for MM and T2 cells; Gibco-Life Technologies, Rockville, MD) or Leibovitz’s L-15 (for MDA-MB231; ATCC, Manassas, VA) media supplemented with 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco-Life Technologies).
Leibovitz s l 15
Manufactured by American Type Culture Collection
Sourced in United States
Leibovitz's L-15 is a cell culture medium developed by American Type Culture Collection (ATCC). It is designed to support the growth and maintenance of a variety of cell types in vitro. The medium provides the necessary nutrients and growth factors for cell proliferation and viability.
Automatically generated - may contain errors
Lab products found in correlation
Mda mb 231, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by American Type Culture Collection (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Hpaf 2, by American Type Culture Collection (2 mentions)
Cfpac, by American Type Culture Collection (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by American Type Culture Collection (1 mentions)
Hsb 2, by American Type Culture Collection (1 mentions)
Mm 1s, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Hydrocortisone, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by STEMCELL (1 mentions)
Human insulin, by Merck Group (1 mentions)
Cholera toxin, by Thermo Fisher Scientific (1 mentions)
Mccoy s 5a medium, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
8 protocols using leibovitz s l 15
1
Cultivation of Diverse Cell Lines
2
Comprehensive Cell Line Characterization
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Human PCa cell lines (PC3, DU145, LNCaP and its invasive subclone LNCaP LN3) and prostate epithelial cells (PreC) along with two breast cancer cell lines (MDA-MB-468 and MDA-MB-231) were used in various in vitro studies. All cells were purchased from American Type Culture Collection (ATCC). Cells were maintained in F-12K (ATCC), Eagle’s Minimum Essential Medium (EMEM; ATCC), Roswell Park Memorial Institute (RPMI) 1640 (ATCC), or Leibovitz’s L-15 (ATCC) cell-culture medium, according to the culture method for each cell type per the instructions from ATCC, supplemented with high-glucose, 10% fetal bovine serum (FBS; Gibco®) and 1% penicillin/streptomycin antibiotic (Thermo-Fisher Scientific). Cell culture and all biological experiments were performed at 37°C in 5% CO2 conditions in a cell-culture incubator. All cells were authenticated (using the “DDC Biomedical” or “Genetica DNA Laboratories” cell line authentication test) and checked for mycoplasma contamination before in vitro cell experiments and in vivo xenograft tumour model preparation.
3
Cell Line Culture Protocols
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Human embryonic kidney cell line HEK293T, human non-tumorigenic epithelial cell line MCF10A and human breast cancer cell lines Hs578T, MDA-MB231 and SKBR3 and HeLa cell line were obtained and cultured as recommended by American Type Culture Collection (ATCC, Gaithersburg, MD, USA). MCF7, BT549 and T47D were provided from Alex Toker’s lab (BIDMC, USA). HEK293T and HeLa were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). MCF10A was maintained in F12 medium (DMEM-F12, Gibco, Carlsbad, CA, USA) supplemented with 5% horse serum, hydrocortisone (Invitrogen, Carlsbad, CA, USA), insulin (0.01 mg/mL, Sigma-Aldrich, St. Louis, MO, USA), epidermal growth factor (20 ng/mL, NJ, USA), hydrocortisone (0.5 mg/mL, Stemcell technology, Canada), cholera toxin (100 ng/mL) and penicillin–streptomycin (100 μg/mL each, Invitrogen, Carlsbad, CA, USA). MCF7 and Hs578T were maintained in DMEM supplemented with 10% FBS and human insulin (0.01 mg/mL, Sigma-Aldrich, St. Louis, MO, USA). BT549 and T47D were maintained in Roswell Park Memorial Institute 1640 (RPMI1640, Gibco, Carlsbad, CA, USA) with 10% FBS. MDA-MB231 was maintained in Leibovitz's L15 (ATCC, Gaithersburg, MD, USA) supplemented with 10% FBS. SKBR3 was maintained in McCoy's 5a Medium (ATCC, Gaithersburg, MD, USA) with 10% FBS.
4
Cancer Cell Lines: Cultivation and Maintenance
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The 54 cancer cell lines used in this study consisted of 11 wild-type (4 urothelial cancer, 5 melanoma, and 2 lung cancer), 2 with structural rearrangement (1 melanoma, 1 glioblastoma), and 41 mutant (20 melanoma and 21 urothelial cancer) cell lines (Supplemental Table S1 ). WM-39, WM-46, WM-51, WM-88, and WM-4002 (Rockland Antibodies and Assays; Gilbertsville, PA, USA) were grown in Tu2% media (80% MCDB-153 (Sigma-Aldrich; St. Louis, MO, USA), 20% Leibovitz’s L-15 (ATCC; Manassas, VA, USA), 5 µg/mL bovine insulin (Sigma-Aldrich; St. Louis, MO, USA), 1.68 mM CaCl2 (Sigma-Aldrich; St. Louis, MO, USA), and 2% FBS (Peak Serum; Wellington, CO, USA)). NCI-H358 and NCI-H2122 cells (ATCC; Manassas, VA, USA) were grown in RPMI-1640 (ThermoFisher; Waltham, MA, USA) with 10% FBS. LN-18 cells (ATCC; Manassas, VA, USA) were grown in DMEM (ThermoFisher; Waltham, MA, USA) with 5% FBS. 253J cells (MD Anderson) were grown in MEM, 0.1 mM non-essential amino acids (ThermoFisher; Waltham, MA, USA), 1 mM sodium pyruvate (ThermoFisher; Waltham, MA, USA) with 10% FBS. UMUC3 cells (ATCC; Manassas, VA, USA) were grown in MEM (ThermoFisher; Waltham, MA, USA), 1 mM sodium pyruvate, and 10% FBS. VMCUB3 cells were grown in MEM, 0.1 mM non-essential amino acids, and 1 mM sodium pyruvate with 10% FBS.
5
Laser Microdissection of Cultured Cell Lines
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Pancreatic cancer cell lines, HPAF-II (CRL-1997) and CFPAC (CRL-1918), T lymphocyte cell line, Jurkat (TIB-152), and breast cancer cell like, MDA-MB-231 (HTB-26), were obtained from American Type Culture Collection (ATCC), and grown using EMEM (HPAF-II), IMDM (CFPAC), RPMI 1640 (Jurkat) and Leibovitz’s L-15 (MDA-MB-231) medium (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 100 U/mL penicillin-streptomycin (ATCC). All cell lines were grown at 37°C with 5% CO2 and were routinely passaged at 80% confluence using an iso-osmotic sodium citrate solution for cell release (Thermo). When preparing cells for laser microdissection, cells were released from the culture plates using the same sodium citrate solution. Following a wash with the culture medium, each cell line was diluted to a density of 1000 cells per 100 μL. Approximately 1000 cells (100 μL) were smeared on PEN membrane slides (Leica), air-dried for 10 minutes, and fixed with 100 μL of 100% Ethanol. Cells were then isolated by laser microdissection as outlined below. DNA was extracted using the Qiagen Blood and Cell Culture DNA Mini Kit according to the manufacturer’s protocol. For extraction of DNA from < 100 cells, cell lysis solution from Qiagen RepliG Single Cell kit was used according to the manufacturer’s protocol.
6
Comprehensive Cell Line Characterization
7
Culturing Mammary Carcinoma Cell Lines
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The 4T1‐eGFP‐Puro (Imanis Life Sciences) mammary carcinoma cell line was cultured in RPMI‐1640 media (Quality Biological) supplemented with 10% heat‐inactivated exosome‐free foetal bovine serum (FBS) (Peak Serum), 2 mM L‐glutamine (Quality Biological), 100 ug/ml streptomycin (Quality Biological), 100 U/ml penicillin (Quality Biological), and 2 ug/ml puromycin (Invivogen). The MDA‐MBA‐231 (ATCC) adenocarcinoma cell line was cultured in Leibovitz's L‐15 (ATCC) media supplemented with 10% heat‐inactivated exosome‐free foetal bovine serum (FBS) (Peak Serum), 2 mM L‐glutamine (Quality Biological), 100 ug/ml streptomycin (Quality Biological), and 100 ug/ml penicillin (Quality Biological) for 5 days in a flask (at 37°C and 5% CO2) before harvesting for downstream experiments. For the SLN EV immunogen priming model, the 4T1 (ATCC) mammary carcinoma cell line was cultured in RPMI‐1640 (Quality Biological). When reaching confluency, cells were removed by scraping and resuspended in media after cell viability was determined by Trypan Blue exclusion.
8
Cell Culture and Transfection Techniques
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HCT116 cells (ATCC) were cultured in McCoy’s 5A (ATCC) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 °C with 5% CO2. SW620 cells (ATCC) were cultured in Leibovitz's L-15 (ATCC) and supplemented with 10% FBS at 37 °C with 0% CO2. Cells were transfected with plasmids (400 ng/ml) or siRNA (100 nM) at 70% confluency, using Lipofectamine 3000 (Invitrogen). For experiments that used both siRNA and plasmid, siRNA was transfected first, and the medium was replaced with fresh before the plasmid was transfected on the following day. For TOP/FOPFLASH assays, cells were transfected with TOPFLASH or FOPFLASH construct for 24 h and luciferase assays were carried out as described previously (18 (link)). For cleavage assays, cells were transfected and subsequently cultured in serum-free medium for 48 h (18 (link)), and Western blotting was performed as described below.
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