C57BL/6J (wild type) mice (stock number: 000664), C3−/− mice (N7, Stock No: 003641), TCRα−/− mice (N13, Stock No: 002116), Il10−/− mice (N13, Stock No: 002251), Il10 GFP reporter (Tiger) mice (N10, stock number: 008379) were purchased from The Jackson Laboratory (Bar Harbor, ME). C3−/− mice were further backcrossed to C57BL/6J for 5 generations (N12). N indicates the number of backcrossed generations. Six to eight week old mice were used for all experiments. All mice strains are of the C57BL/6 genetic background. Mice were housed in a specific pathogen-free facility in the Duke University Medical Center and used according to protocols approved by the Duke University Institutional Animal Care and Use Committee. The B16-F10 murine melanoma was purchased from ATCC in 2011. E0771, a murine mammary adenocarcinoma, was a gift from Dr. Scott A. Gerber (University of Rochester) in 2013. The mouse tumor cell lines used in this manuscript have not been authenticated by us.
B16f10 murine melanoma
Manufactured by American Type Culture Collection
Sourced in United States
B16F10 murine melanoma is a cell line derived from a melanoma tumor in a C57BL/6 mouse. It is a commonly used model for studying melanoma in preclinical research.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Wild type c57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Mcf 7 breast, by American Type Culture Collection (1 mentions)
Htb 72, by American Type Culture Collection (1 mentions)
Acc 107, by Leibniz Institute DSMZ (1 mentions)
Sk mel 28, by American Type Culture Collection (1 mentions)
Hek blue tlr4 reporter cells, by InvivoGen (1 mentions)
Female balb c mice, by Jackson ImmunoResearch (1 mentions)
Female c57bl 6 mice, by Charles River Laboratories (1 mentions)
Chemstrips, by Roche (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4t1 murine mammary carcinoma, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Nacalai Tesque (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Non essential amino acids (neaa), by Nacalai Tesque (1 mentions)
11 protocols using b16f10 murine melanoma
1
Murine Tumor Models for Immunology
2
Anticancer Activity Evaluation of Compounds
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Compounds 1 –8 were evaluated for their in vitro growth inhibitory activity against five human tumor cell lines, including the A549 non-small cell lung cancer (ACC107, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), the U373 glioblastoma (ECACC 08061901, European Collection of Authenticated Cell Cultures, Salisbury, UK), the Hs683 oligodendroglioma (ATCC HTB-138, American Type Culture Collection, Manassas, VA, USA), the MCF7 breast cancer (ATCC HTB22) and the SKMEL28 melanoma (ATCC HTB72) cell lines, and against the B16F10 murine melanoma (ATCC CRL 6475) cell line. The inhibitory growth activity of the compounds under study was determined by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay [34 (link),35 (link)]. The cancer cells were cultured during three days in the presence of the compounds and the data were reported as mean IC50 values calculated on the sextuplicates of the experiment conducted once for each compound and for each cell line.
3
Cell Lines for Mammary Carcinoma and Melanoma
4
Comparative Cell Culture Protocol
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B16F10 murine melanoma, A375 and SKMel28 human melanoma cells were purchased from ATCC (VA, USA) and maintained in our in house repository. Non-cancerous cells, AML12 (mouse hepatocytes), L6 (rat muscle cells) and MEF (mouse embryonic fibroblasts) were used in parallel along with cancer cells as a control. All the cells were grown in their respective medium containing either 1 mM or 25 mM glucose depending upon the experiments and supplemented with 10% heat inactivated fetal bovine serum (Hyclone, UT, USA), penicillin (100 U/ml) and streptomycin 100 μg/ml (Life Technologies, NY, USA), at 37°C in presence of 5% CO2.
5
Murine Tumor Models for Immunotherapy
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Female C57BL/6 mice (aged
8–12 weeks) were purchased from Charles River Laboratory. Female
Balb/c mice (aged 8–12 weeks) were purchased from Jackson Laboratory.
B16F10 murine melanoma, CT26 and MC38 murine colon carcinoma, EMT6
murine mammary carcinoma, and MDA-MB-231 human breast adenocarcinoma
cell lines were purchased from ATCC and cultured according to instructions,
with routine checks for mycoplasma contamination. Tumor inoculations
were 500 000 cells in 30 μL of sterile PBS unless otherwise
noted. Polymer solutions were verified as endotoxin-free prior to
injection via HEK-Blue TLR4 reporter cells (InvivoGen). Tumor dimensions
were measured with digital calipers, and volume was calculated as
height × width × thickness × (π/6). All of the
animal experiments performed in this research were approved by the
Institutional Animal Care and Use Committee of the University of Chicago
under protocol 72456.
8–12 weeks) were purchased from Charles River Laboratory. Female
Balb/c mice (aged 8–12 weeks) were purchased from Jackson Laboratory.
B16F10 murine melanoma, CT26 and MC38 murine colon carcinoma, EMT6
murine mammary carcinoma, and MDA-MB-231 human breast adenocarcinoma
cell lines were purchased from ATCC and cultured according to instructions,
with routine checks for mycoplasma contamination. Tumor inoculations
were 500 000 cells in 30 μL of sterile PBS unless otherwise
noted. Polymer solutions were verified as endotoxin-free prior to
injection via HEK-Blue TLR4 reporter cells (InvivoGen). Tumor dimensions
were measured with digital calipers, and volume was calculated as
height × width × thickness × (π/6). All of the
animal experiments performed in this research were approved by the
Institutional Animal Care and Use Committee of the University of Chicago
under protocol 72456.
6
Maintaining and Inoculating Murine Cancer Cell Lines
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The CT26 murine colon cancer and B16F10 murine melanoma cell lines (ATCC, Manassas, VA, USA) were maintained in RPMI 1640 medium (Thermal Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 1% streptomycin/penicillin at 37°C in an incubator with 5% CO2. On day of inoculation (day 1), the cells were detached using 5% trypsin for 5 minutes and washed once with phosphate-buffered saline (PBS). The cells were then resuspended in PBS and counted using a hematocytometer.
7
Induction of Melanoma-Specific Diabetes in Mice
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Six- to 8-week-old C57BL/6 mice (WT) were obtained from National Cancer Institute/Charles River program. RIP-gp mice were obtained from P. Ohashi (Princess Margaret Cancer Center, University of Toronto, Toronto, Canada) and bred in our facility. B16F10 murine melanoma was obtained from the American Type Culture Collection, Manassas, VA. B16F10 cells transfected with a mini-gene plasmid encoding the LCMV gp33-41 CD8 T cell epitope (B16F10gp33-41) were made by A. Prevost-Blondel, et al., [20 (link)], were kindly provided by P. Ohashi and maintained in selection media containing 200 μg/ml G418. Blood glucose levels were measured 2–3 times per week using Accuchek III Glucometers and Chemstrips (Roche) and mice were considered diabetic following 2 consecutive measurements > 250 mg glucose/dl.
8
Murine Cancer Cell Lines and Macrophage Derivation
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B16F10 murine melanoma, 4T1 murine mammary carcinoma, and 293T human embryonic kidney cell lines were all purchased from the American Type Culture Collection (ATCC). Luciferase-tagged B16F10 and 4T1 cells were established by transfection of B16F10 and 4T1 cells with vectors carrying luciferase and puromycin resistance gene. For construction of SIRPα variant-engineered cells, the cells were sorted and sub-cloned after being transduced by lentivirus expressing cell membrane bound SαV (SαV is an engineered high-affinity SIRPα variant fused with murine SIRPα transmembrane domain). The cells were cultured in 5% CO2 and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Invitrogen). Bone marrow-derived macrophages (BMDMs) were prepared following the steps blow. C57BL/6 or BALB/c mice were sacrificed and bone marrow cells were isolated from leg bones, maintained in RPMI medium supplemented with 10% FBS and 1% antibiotics, and differentiated with macrophage colony-stimulating factor (M-CSF) for 7 days.
9
Generation of Tumor-Specific T Cell Models
10
Cell Line Culturing for Preclinical Research
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B16F10 murine melanoma, 4T1 mammary carcinoma, and DU145 (androgen-independent human prostate) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). B16BL6 murine melanoma cell line was a gift from Dr. Yoshio Okada. Human embryonic kidney (293FT) cells were purchased from Thermo Fisher Scientific. B16F10 and DU145 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Nacalai Tesque Inc., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France), 100 U/mL penicillin and 0.1 mg/mL streptomycin (penicillin–streptomycin Mixed solution) (Nacalai Tesque). 4T1 and B16BL6 cells were maintained in RPMI-1640 medium (Nacalai Tesque) supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. 293FT cells were maintained in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate (Nacalai Tesque), 2 mM l-glutamine (Nacalai Tesque), and 1% nonessential amino acids (Nacalai Tesque). G418 (500 μg/mL) was added to a subculture of 293FT cells. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
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