Prostates were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 5 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF, 1× complete protease inhibitors), boiled at 100°C for 10 min in Laemmli buffer (BioRad) with 2-mercaptoethanol, and run on a 4–20% gradient SDS-PAGE gel (BioRad). Following transfer to a nitrocellulose membrane, blots were probed with rabbit anti-mouse mAbs to CREB or pCREB (Cat #9197S and 9198, respectively, Cell Signaling), rabbit anti-mouse polyclonal Ab to pPKA C (Cat #4781S, Cell Signaling) or rabbit anti-mouse polyclonal IgG to β-tubulin (Cat #2146S, Cell Signaling) followed with mouse anti-rabbit HRP conjugate (Cat #SC-2357, Santa Cruz), and developed using an ECL plus chemiluminescence kit (Cat#32132, Thermo Fisher Scientific). B16-F10 melanoma cells (Cat#CRL-6475, ATCC) treated 1 h with or without 25 μM forskolin (FSK, adenylyl cyclase activator) served as pCREB positive and negative controls, respectively. Protein quantification was performed using GeneTools software (Syngene).
B16f10 melanoma cells
Manufactured by American Type Culture Collection
Sourced in United States
B16F10 melanoma cells are a well-established cell line derived from a mouse melanoma. They are widely used in cancer research for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Sodium pyruvate, by Merck Group (4 mentions)
Non essential amino acids (neaa), by Merck Group (4 mentions)
Hepes, by Merck Group (4 mentions)
Pen strep l glutamine, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
4t1 mammary carcinoma cells, by American Type Culture Collection (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Ecl plus chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Laemmli buffer, by Bio-Rad (2 mentions)
Genetools software, by Syngene (2 mentions)
Gradient sds page gel, by Bio-Rad (2 mentions)
Rabbit anti mouse mabs to creb or pcreb, by Cell Signaling Technology (2 mentions)
Rabbit anti mouse polyclonal ab to ppka c, by Cell Signaling Technology (2 mentions)
100 protocols using b16f10 melanoma cells
1
Prostate CREB Activation Analysis
2
Stable Transfection of Murine NIS in B16F10 Melanoma and DHD/K12/TRb Colorectal Cancer Cells
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The B16F10 melanoma cells (obtained from ATCC) and the DHD/K12/TRb colorectal cancer cells (obtained from Sigma) were cultured in DMEM (GIBCO, France) supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO) at 37°C under a humidified atmosphere containing 5% CO2. Cells were passaged by using 0.05% trypsin. Both cell lines were transfected with pcDNA3.1-mNIS (murine NIS)36 (link) using the FuGENE 6 reagent (Roche) according to the manufacturer’s instructions. Stable clones were selected by addition of 1 mg/mL geneticin (G418) to the medium 3 days after transfection. One clone with high functional expression of NIS was selected for each cell line.37 (link)
3
Murine Models for Cancer Research
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C57Bl/6J (B6) WT mice and Myc-GFP mice (B6;129-Myctm1Slek/J) were purchased from Charles River and Jackson Laboratories. PLZF-GFP (Zbtb16GFP) mice were generated in the laboratory of D. Sant’Angelo as described previously51 (link). Mice were maintained under specific pathogen-free conditions. Standard food and water were given ad libitum. Where indicated, mice were fed high fat diet (HFD) (Research Diets; D12492) for 8 weeks. Mice were used at the foetal (embryonic day 14-18), neonatal (1-5 days old) or adult (6-12 weeks old) stages.
The E0771 murine breast adenocarcinoma cells, MC38 murine colon adenocarcinoma cells and B16.F10 melanoma cells were purchased from ATCC (Manassas, VA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (vol/vol) FCS (Gibco; Thermo Fisher Scientific) and 1% (vol/vol) penicillin/streptomycin (Sigma-Aldrich).
The E0771 murine breast adenocarcinoma cells, MC38 murine colon adenocarcinoma cells and B16.F10 melanoma cells were purchased from ATCC (Manassas, VA). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (vol/vol) FCS (Gibco; Thermo Fisher Scientific) and 1% (vol/vol) penicillin/streptomycin (Sigma-Aldrich).
4
Culturing B16F10 and LLC Cells
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B16F10 melanoma cells and LLC cells were purchased from ATCC (Manassas, VA, USA). These cells were cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10% fetal calf serum and 1% penicillin and streptomycin. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2.
5
CD47-Dependent Tumor Irradiation Response
6
Culturing B16F10 Melanoma Cells
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B16F10 melanoma cells were purchased from ATCC (CRL-6475) and maintained in RPMI with 10% FCS, 1% Pen/Strep/L-glutamine (Sigma), 1% non-essential amino acids (Sigma), 1% sodium pyruvate (Sigma), 10 mM HEPES (Sigma) and 0.1 mM β-mercaptoethanol.
7
Evaluating DTIC Efficacy on B16-F10 Melanoma
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B16-F10 melanoma cells were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, containing 10% fetal bovine serum (FBS). Before cells were seeded into 96-well plates, we determined cell viability primarily by trypan blue staining and determined the number of cells by cell counting. The percentage of viable cells was about 99% and the cell number was about 3×104 cells/mL. Cells in exponential growth were placed in 96-well plates at a final concentration of 3×104 cells/mL and then treated with differing concentrations of DTIC for differing periods of time. Cell viability was assessed using the Cell Counting Kit (CCK-8) assay (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The absorbance of each well was read at 490 nm with MK3 Absorbance Microplate Reader (Thermo Electron Corporation, Beverly, MA, USA). All data were derived from three independent experiments. Cell inhibition rates (%) were determined as follows: 1− [ODtreatment − ODblank/ODcontrol − ODblank] × 100%.
8
Engineered Melanoma and Thymoma Cells
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B16-F10 melanoma cells (ATCC, CRL-6475), MC57 fibrosarcoma cells (ATCC, CRL-2295) and EL4 thymoma cells (ATCC, TIB-39) were obtained from ATCC. B16-F10 and MC57 were cultured in DMEM supplemented with 10% FBS, EL4 cells were cultured in RPMI supplemented with 10% FBS. To generate B16gp33 and EL4gp33, B16-F10 and EL4 were transduced with pHRST-IRES-eGFP lentiviral vector modified to express gp33 peptide, and FACS-sorted for GFP-positive population.
9
Tumor Growth and Immunophenotyping in Mice
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All animal procedures were in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of Washington State University. 8- to 12-week old male wild-type and Per1/2−/− mice on C57BL/6 background [36 (link)] were obtained from the Jackson Laboratories. The mice were maintained under a 12 h/12 h light/dark cycle (light on at 7 AM, ZT0, and off at 7 PM, ZT12) at least 4 weeks before and through the duration of the study.
For tumor studies, B16F10 melanoma cells were purchased from ATCC and cultured in RPMI-1640 + 10% FBS. 2×105 cells in 50% matrigel (Corning) were injected into the lower right flank region of each mouse. Body weights were measured using an analytical balance, and tumor volumes were measured using a digital caliper and calculated using the formula: V = (W2 x L)/2 [63 (link)]. Tumor-bearing mice were sacrificed as tumor volumes crossed 4X the volume at the start of cisplatin treatment (for toxicity and tumor study) or reached an average of 650 mm3 on day 15 (for immunophenotyping). Upon sacrifice, blood, kidney, spleen, lymph nodes, testis, brain, and tumor tissues were harvested for further analysis.
For tumor studies, B16F10 melanoma cells were purchased from ATCC and cultured in RPMI-1640 + 10% FBS. 2×105 cells in 50% matrigel (Corning) were injected into the lower right flank region of each mouse. Body weights were measured using an analytical balance, and tumor volumes were measured using a digital caliper and calculated using the formula: V = (W2 x L)/2 [63 (link)]. Tumor-bearing mice were sacrificed as tumor volumes crossed 4X the volume at the start of cisplatin treatment (for toxicity and tumor study) or reached an average of 650 mm3 on day 15 (for immunophenotyping). Upon sacrifice, blood, kidney, spleen, lymph nodes, testis, brain, and tumor tissues were harvested for further analysis.
10
Culturing Mouse Colorectal Cancer Cell Lines
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Mouse CRC cell line CT26 (ATCC CRL-2638) was purchased from ATCC. Dr. Nicholas Haining kindly provided mouse CRC cell line MC38. MC38 cells stably expressing miR322 inhibitor mouse homolog to miR-424 (MC38-424i) and MC38-miR-control cells were used in this study as described by Zhao et al.19 (link) CT26 cells were cultured in complete Roswell Park Memorial Institute Medium (RPMI) 1640 (Gibco), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen Life Technologies). MC38-WT, MC38-424i, and MC38-miR-control cells were cultured in the complete Dulbecco’s modified Eagle’s medium (DMEM) (Gibco), supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. B16-F10 melanoma cells (ATCC CRL-6475) were purchased from ATCC and were cultured in the complete DMEM (Gibco), supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Cell lines were authenticated and routinely tested for mycoplasma.
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