B16 f10 melanoma

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B16-F10 melanoma is a cell line derived from a mouse melanoma tumor. It is commonly used in cancer research to study tumor growth, metastasis, and response to various treatments.

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36 protocols using b16 f10 melanoma

Cell Culture and Cytotoxicity Assays

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Both mouse EL-4 lymphoma and B16F10 melanoma cells were acquired from ATCC (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM, Corning, Corning, NY, USA). CT26 colon carcinoma cells (ATCC) were cultured in RPMI 1640 (Corning). Culture media were supplemented with L-glutamine (ATCC), 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco, Gaithersburgh, MD, USA).
Thaspigargin (1 µM), mitoxantrone (0.5–1 µM), doxorubicin (10–25 µM), and staurosporin (10 µM) were all from Sigma (Sigma-Aldrich, St. Louis, MO, USA).

Rossi A., N. Pakhomova O., Mollica P.A., Casciola M., Mangalanathan U., G. Pakhomov A, & Muratori C. (2019). Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer. Cancers, 11(12), 2034.

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Culturing B16-F10 and CT26 Cells

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B16-F10 melanoma and CT26 colon carcinoma cells were both obtained from ATCC (Manassas, VA, USA). The tumor cells were grown up to a maximum confluence of 80% at 37°C, 5% CO₂, and 95% humidity, in RPMI 1640 (Sigma Aldrich, Munich, Germany) with the addition of 10 % fetal bovine serum (FBS, Biochrom AG, Berlin, Germany) and 1% penicillin-streptomycin (PenStrep, Gibco, Carlsbad, USA).

Seitz C., Rückert M., Deloch L., Weiss E.M., Utz S., Izydor M., Ebel N., Schlücker E., Fietkau R., Gaipl U.S, & Frey B. (2019). Tumor Cell-Based Vaccine Generated With High Hydrostatic Pressure Synergizes With Radiotherapy by Generating a Favorable Anti-tumor Immune Microenvironment. Frontiers in Oncology, 9, 805.

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Cell Line Culture Protocols

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The human cell lines H1650, A549, BEAS2B and HEK293 were obtained from American type culture collection (ATCC) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified 5% CO₂ incubator at 37°C. The mouse NSCLC cell line KP1 was previously generated from lung tumors of KRAS^G12D/+/p53^−/− (KP1) mice (26 (link)). Mouse LLC1 lung cancer cells and B16-F10 melanoma cells were obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 5% CO₂ incubator at 37°C.

Wennerberg E., Mukherjee S., Spada S., Hung C., Agrusa C., Chen C., Valeta-Magara A., Rudqvist N.P., Van Nest S., Kamel M.K., Nasar A., Narula N., Mittal V., Markowitz G., Zhou X.K., Adusumilli P.S., Borczuk A., White T.E., Khan A.G., Balderes P., Lorenz I.C., Altorki N., Demaria S., McGraw T.E, & Stiles B.M. (2022). Expression of the mono-ADP-ribosyltransferase ART1 by tumor cells mediates immune resistance in non-small cell lung cancer. Science translational medicine, 14(636), eabe8195.

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Cell Line Characterization and Maintenance

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J774A.1 murine monocytic cell line was acquired in 1997 as a gift from Dr. Stephen A. Stohlman (Cleveland Clinic Foundation, Cleveland, OH). B16-F10 melanoma and Colon 26 adenocarcinoma were acquired in 1988 and 1999, respectively, from ATCC (Manassas, VA). All cell lines were grown in RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), non-essential amino acids, penicillin G and streptomycin (Gemini Bio-Products, West Sacramento, CA). Cell lines were authenticated by STR DNA profiling by DDC Medical (Fairfield, OH) in 2015.

Jang J.K., Khawli L.A., Canter D.C., Hu P., Zhu T.H., Wu B.W., Angell T.E., Li Z, & Epstein A.L. (2016). Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models. Cancer immunology, immunotherapy : CII, 65(5), 511-523.

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Expansion and Cryopreservation of Cell Lines

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The 4 T1 mammary carcinoma and the B16-F10 melanoma cell lines were initially obtained from ATCC and provided to us by Active Biotech. The EG7 cell line (OVA-transfected EL4 lymphoma cell line) [44 (link)] was obtained from Dr Clotilde Thery, Institute Curie, INSERM U932, Paris, France. The cell lines were expanded, frozen in aliquots and new aliquots regularly used for the in vivo experiments. The cells were cultured in RPMI medium (RPMI-1640 supplemented with 10 % fetal calf serum, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin-streptomycin and 50 μM β-mercaptoethanol; all supplements from Invitrogen Life Technologies, Paisley, UK) at 37 °C, 5 % CO₂. For trypsinization of 4 T1 cells, trypsin-EDTA (Sigma-Aldrich, St. Louis, MO) was briefly added to cells at approx. 80 % confluence and the cells were washed with RPMI medium.

Deronic A., Leanderson T, & Ivars F. (2016). The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b+ Ly6Chi cells to tumor tissue reduces tumor growth. BMC Cancer, 16, 440.

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Expansion and Characterization of Cancer Cell Lines

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LLC Lewis lung carcinoma (LL/2, RRID:CVCL_4358), LL/2-Luc2 luciferase expressing Lewis lung carcinoma (referred to as LL2, RRID:CVCL_A4CM), B16F10 melanoma (RRID:CVCL_0159), and EL4 lymphoma (RRID:CVCL_0255) cell lines were purchased from ATCC. Cells were expanded, frozen, and vials were kept in liquid nitrogen. Upon thawing, cells were kept in culture for 4–6 weeks. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO2 and were Mycoplasma negative as detected using Universal Mycoplasma Detection Kit (ATCC).

Deng H., Lin C., Garcia-Gerique L., Fu S., Cruz Z., Bonner E.E., Rosenwasser M., Rajagopal S., Sadhu M.N., Gajendran C., Zainuddin M., Gosu R., Sivanandhan D., Shelef M.A., Nam B., Vogl D.T., Gabrilovich D.I, & Nefedova Y. (2022). A novel selective inhibitor JBI-589 targets PAD4-mediated neutrophil migration to suppress tumor progression. Cancer research, 82(19), 3561-3572.

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Establishing Murine Tumor Models

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A20 B cell lymphoma, B16-F10 melanoma, and CT26 colon carcinoma line were obtained from ATCC (Manassas, VA), 4T1-Luc breast carcinoma cell line was a gift from the S. Strober laboratory and the C. Contag laboratory (both at Stanford University). Tumor cells were cultured in complete medium (RPMI 1640 DMEM- for B16-F10; Cellgro) containing 10% fetal bovine serum (FBS;HyClone), 100 U/mL penicillin, 100μg/mL streptomycin, and 50μM 2-ME (Gibco). Cell lines were routinely tested for mycoplasma contamination.
Six to eight-week-old female BALB/c and C57BL/6 were purchased from Charles River (http://www.criver.com). FVB/N-Tg(MMTV-PyVT)634Mul/J male FVB/NJ females, C57BL/6- Tg(Foxp3-DTR/EGFP)23.2Spar also known as DEREG mice were purchased from JAX Laboratories (http://jaxmice.jax.org/). Mice were housed in the Laboratory Animal Facility of the Stanford University Medical Center (Stanford, CA). All experiments were approved by the Stanford administrative panel on laboratory animal care and conducted in accordance with Stanford University animal facility guidelines.

Sagiv-Barfi I., Czerwinski D.K., Levy S., Alam I.S., Mayer A.T., Gambhir S.S, & Levy R. (2018). Eradication of spontaneous malignancy by local immunotherapy. Science translational medicine, 10(426), eaan4488.

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Evaluating Tumor-Infiltrating Lymphocytes in B16 Melanoma

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B16F10 melanoma (CRL-6475) cell line was purchased from ATCC. 5×10⁵ cells were injected subcutaneously at the flanks of the mice. Tumors were measured in two dimensions by caliper as the product of two perpendicular diameters. TILs were isolated on day 14 to day 20 post tumor implantation as they were reaching 200mm² in size ^{6 (link)}. Single cells suspensions were stained for CD8, CD4, Tim-3, and PD-1. For functional assay, intracellular cytokine staining was conducted as described before ^{6 (link)}. For gene expression analysis, CD8⁺ 7AAD⁻ TILs of B16 melanoma on WT C57BL/c mice or WSX-1^−/− mice were sorted by BD FACSAria after magnetic separation by Dynabeads FlowComp Mouse CD8 (Invitrogen). CD44^hi CD62L^low memory CD8⁺ splenocytes from B6 non-tumor bearing mice were also sorted as a control. RNA from sorted CD8⁺ cells were then extracted and reverse transcribed to cDNA. Gene expressions were quantified by qPCR.

Zhu C., Sakuishi K., Xiao S., Sun Z., Zaghouani S., Gu G., Wang C., Tan D.J., Wu C., Rangachari M., Pertel T., Jin H.T., Ahmed R., Anderson A.C, & Kuchroo V.K. (2015). An IL-27/NFIL3 signaling axis drives Tim-3 and IL-10 expression and T cell dysfunction. Nature communications, 6, 6072.

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Tumor Immunology: Treg Modulation

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B16F10 melanoma cells were obtained from ATCC (CRL-6475, Manassas, VA). MC38 colon adenocarcinoma cells were purchased from Kerafast (CVCL_B288, Boston, MA). MC38 and B16F10 cells were cultured in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% FCS (Life Technologies), 1% Penicillin-Streptomycin, 1% L-Glutamine, and 1% HEPES. A total of 1 × 10⁵ tumor cells was suspended in 100 μl PBS and inoculated subcutaneously into the right hind flank of WT (Foxp3^Cre) or Foxp3^CreHif2a^f/f mice. Tumor size was determined by measuring tumor length and width, using volume as readout. Volumes (V) were calculated using the equation: V = L × W²/2, where L is the long diameter and W is the short diameter. To evaluate the effect of HIF-2α-suppressed iTreg cells, C57BL/6 mice were lightly irradiated (2 Gy), followed by subcutaneous transplantation with 5 × 10⁵ MC38 cells. Sorted CD4⁺CD25⁺ iTreg cells were untreated or treated with 20 μM of the HIF-2α-specific inhibitor PT2385 (HY-12867, Medchem Express, Monmouth Junction, NJ) for 16 h and then adoptively transferred into mice on days 3, 6 and 10 after tumor implantation.

Hsu T.S., Lin Y.L., Wang Y.A., Mo S.T., Chi P.Y., Lai A.C., Pan H.Y., Chang Y.J, & Lai M.Z. (2020). HIF-2α is indispensable for regulatory T cell function. Nature Communications, 11, 5005.

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Dietary Sodium Modulates Tumor Growth in Mice

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Mice were either fed a normal diet (Control group) containing 0.5% NaCl or sodium enriched diet (HSD group) containing 4% NaCl as well as 1% NaCl enriched tap water for 2 weeks before tumor inoculation. In some experiments, diet switch was started directly before tumor inoculation. Both diets were purchased from SSNIFF (Ctrl: E15430-04, HSD: E15431-34; Soest, Germany). Mice were maintained on the respective diet during the course of the experiment. B16F10 melanoma cells (ATCC) were cultured in DMEM (Sigma Aldrich) supplemented with 10% FCS (Gibco) and Penicillin/Streptomycin (Gibco) and Lewis Lung carcinoma (LLC) cells (ATCC) were cultured in RPMI (Lonza) supplemented with 10% FCS and Penicillin/Streptomycin. Cells were maintained mycoplasma free, tested continuously by HEK-blue mycoplasma detection (Invivogen). Tumor cells were subcutaneously injected in the left abdominal flank (LLC at 1 × 10e6 expose 6 and B16F10 at 2 × 10e5 expose 5 cells/mouse). Tumor growth was monitored three times per week by using a caliper. Tumor volume was calculated with the ellipsoid formula (π/6^*a^*b^*c) as described before (26 (link)).

Willebrand R., Hamad I., Van Zeebroeck L., Kiss M., Bruderek K., Geuzens A., Swinnen D., Côrte-Real B.F., Markó L., Lebegge E., Laoui D., Kemna J., Kammertoens T., Brandau S., Van Ginderachter J.A, & Kleinewietfeld M. (2019). High Salt Inhibits Tumor Growth by Enhancing Anti-tumor Immunity. Frontiers in Immunology, 10, 1141.

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