Human melanoma cells SK-MEL-28, SK-MEL-2, and SK-MEL-5 (ATCC) and mouse cell line CT26 (ATCC) were cultured in monolayers using RPMI supplemented with 10% heat inactivated bovine serum (Thermo Fisher Scientific), 10mM L-glutamine (Corning), and 0.5% penicillin G-streptomycin sulfate (Corning). Cells were detached using 0.25% trypsin EDTA (Corning) for passaging. The murine melanoma cell line D4M3A was generated from Tyr::CreER;BrafCA;Ptenlox/lox mice (13 (link)) and kindly provided by Dr. David Mullins (Dartmouth University, Hanover, NH). D4M3A cells were cultured as previously described (13 (link)). All cells were low-passage and confirmed to be mycoplasma-free (LookOut mycoplasma kit; Sigma).
Heat inactivated bovine serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Heat-inactivated bovine serum is a laboratory product derived from the blood of bovine sources. It is a complex mixture of proteins, growth factors, and other components that is commonly used in cell culture media to support the growth and maintenance of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Corning (2 mentions)
Penicillin g streptomycin sulfate, by Corning (2 mentions)
Lookout mycoplasma kit, by Merck Group (2 mentions)
Trypsin edta, by Corning (2 mentions)
2 7 dichlorofluorescein dcfh, by Merck Group (2 mentions)
Bis trispolyacrylamide gels, by Bio-Rad (2 mentions)
Sypro ruby protein gel stain, by Bio-Rad (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Lds loading buffer, by Thermo Fisher Scientific (2 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Gel doc xr, by Bio-Rad (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sk mel 28, by American Type Culture Collection (1 mentions)
Mts cell proliferation colorimetric assay kit, by Abcam (1 mentions)
Sk mel 2, by American Type Culture Collection (1 mentions)
Sk mel 5, by American Type Culture Collection (1 mentions)
16 protocols using heat inactivated bovine serum
1
Culturing Human and Murine Melanoma Cells
2
Immunofluorescence Staining Protocol
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Ethanol was obtained from SAV LP Co., Ltd. (Flintsbach am Inn, Germany); non-buffered formalin and xylene from Carl Roth Co., Ltd. (Karlsruhe, Germany); Dulbecco’s Modified Eagle’s Medium (DMEM, 1 g/L D-glucose) and 2% heat-inactivated bovine serum from Thermo Fisher Scientific (Dreieich, Germany); and DAPI (4′, 6-diamino-2-phenylindole), bovine serum albumin (BSA), and Tween 20 from Sigma-Aldrich (Deisenhofen, Germany). Details of the primary antibodies used in this study are provided in Table 2 . Goat anti-rabbit IgG Alexa Fluor 488 (1:300; Invitrogen by Thermo Fisher Scientific, Dreieich, Germany) was used as the secondary antibody for indirect immunofluorescence staining.
3
Melanoma Cell Culture and Viability Assay
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Human melanoma cell lines SK-MEL-2, SK-MEL-5, SK-MEL-28, M14, and LOX-IMVI were obtained from ATCC (Manassas, VA). Cells were cultured in monolayers using RPMI supplemented with 10% heat-inactivated bovine serum (Thermo Fisher Scientific), 10mM L-glutamine (Corning), and 0.5% penicillin G-streptomycin sulfate (Corning). Cells were detached using 0.25% trypsin EDTA (Corning) for passaging and were cultured at 37°C in 5% CO2. The murine melanoma cell line D4M3A was generated from Tyr::CreER; BrafCA; Ptenlox/lox mice22 and kindly provided by Dr. David Mullins (Dartmouth University, Hanover, NH). D4M3A cells were cultured as described in Jenkins et al., 2014. All cells used in experiments were low-passage and were confirmed to be mycoplasma-free (LookOut mycoplasma kit; Sigma). During cell viability assays, cells were plated in 96-well plates and treated with T-VEC after 12–16 hr. Cell viability was measured using MTS assay and cell viability measured according to the manufacturer’s instructions (MTS Cell Proliferation Colorimetric Assay Kit, Biovision, Milpitas, CA).
4
Isolation and Analysis of Hematopoietic Progenitors
5
HepG2 Hepatocyte Cell Culture Protocol
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Human liver carcinoma cell line HepG2 and its derivative HBV reporter line HepG2.2.15 (kind gift of Dr. S. Jameel, International Center for Genetic Engineering & Biotechnology, New Delhi, India). Cells were grown and maintained in RPMI-1640 medium (Gibco, USA), supplemented with 10% heat-inactivated bovine serum (Gibco, USA), 1xpenicillin-streptomycin mix, and 1x sodium pyruvate (HyClone Laboratories, USA) at 37 0C in a humified chamber with 5% CO2 supply. 2,7-Dichlorofluorescein (DCFH; Sigma, USA) was used as an inducer of in vitro hepatotoxicity. The approved nucleoside analog-based anti-HBV drug, lamivudine (3TC; Sigma, USA), was used as standard.
6
Comparative Proteomic Analysis of Stem/Progenitor Cells
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Equal numbers of cells from each stem or progenitor population were sorted into HBSS supplemented with 2% (v/v) heat-inactivated bovine serum (GIBCO). Samples were washed once with PBS and centrifuged at 2500 g at 4°C for 5 min. Supernatant was discarded and protein pellets were directly lysed and solubilized in 9M urea, 2% SDS, 50 mM DTT, and 50 mM Tris pH 7.4. After incubating the samples for 10–15 min at room temperature, LDS loading buffer (Life Technologies) was added and the sample was heated at 70°C for 10 minutes. Samples were separated on Bis-Trispolyacrylamide gels (Life Technologies). The Bis-Trispolyacrylamide gels were then stained with SYPRO Ruby Protein Gel Stain (Bio-Rad) for 3 h and washed in a solution containing 7% acetic acid and 10% methanol for 1 h. Gels were imaged using Gel Doc XR (Bio-Rad) and analyzed with Image Lab 6.0.1 software (Bio-Rad).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
7
Isolation of Bone Marrow and Cord Blood Cells
8
Quantification of HSC and HPC in Transplant Mice
9
HepG2 Cell Line Maintenance Protocol
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The human hepatoblastoma cell line, HepG2 (22 (link)) was maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% heat-inactivated bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 1X penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 1X sodium pyruvate (GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified chamber containing 5% CO2. Silymarin, 2,7-dichlorofluorescein (DCFH), ascorbic acid (all Sigma-Aldrich; Merck KGaA) and gallic acid (Fluka; Honeywell International Inc., Morris Plains, NJ, USA) were also purchased.
10
Cell Viability Assay with HIHE Soxhlet
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The T-lymphoblastic cell line CCRC-CEM and human colon adenocarcinoma cell line DLD1 were purchased from LGC Standard (LGC Group, Middelsex, UK). Both cell lines were grown in Roswell Park Memorial Institute (RPMI) 1640 (Sigma Aldrich), supplemented with 10% heat-inactivated bovine serum (Gibco, Life Technologies, Monza, Italy), 1% penicillin/streptomycin solution, 1% l-glutamine solution and, only for CCRC-CEM, with 1% sodium pyruvate solution (all obtained from Biochrome, Merck Millipore, Darmstadt, Germany). Cells were incubated at 37 °C with 5% CO2. To maintain exponential growth, CCRC-CEM were cultured at the density of 2 × 106 mL. DLD1 were trypsinized when they reached 70–80% confluency.
Cells were treated with increasing concentration of HIHE soxhlet (0–100 μg/mL) for 1, 3, 6, or 24 h according to experimental requirements. Etoposide 10 µg/mL, doxorubicin 5 µM, camptothecin 5 µM, H2O2 1 mM, and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) 50 µM were used as positive controls.
Cells were treated with increasing concentration of HIHE soxhlet (0–100 μg/mL) for 1, 3, 6, or 24 h according to experimental requirements. Etoposide 10 µg/mL, doxorubicin 5 µM, camptothecin 5 µM, H2O2 1 mM, and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) 50 µM were used as positive controls.
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