Isolation of mononuclear myogenic cells from adult tibialis anterior muscle was performed as described previously (Murphy et al., 2014 (link)). Tibialis anterior muscles were dissected, minced, and digested for 1 hr at 37°C in 100 μl of 5 mg/ml liberase (Sigma-Aldrich, 5401127001) and 25 μl of 10 U/µl DNAseI (Sigma-Aldrich, 4716728001) in 3 ml Ham’s F12 media (Thermo Fisher Scientific, 11765054). Samples were passed through 70 μm and 40 μm filters, centrifuged at 1800 rpm for 10 min, aspirated supernatant, and pellet resuspended in satellite cell growth media (15% horse serum [Gibco, 16050-122], 1:1000 50 mg/ml gentamicin [Thermo Fisher Scientific, 15750060] in F12 media). Myogenic mononuclear cells were isolated and sorted via GFP on Propel Labs Avalon (Bio-Rad).
Ham s f12 media
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Ham's F12 media is a widely used cell culture medium formulated to support the growth and maintenance of various cell types, including mammalian and insect cells. It provides the necessary nutrients, vitamins, and other components required for optimal cell proliferation and survival in an in vitro environment.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions)
Hydrocortisone, by Merck Group (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Insulin, by Merck Group (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Hepes, by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (4 mentions)
Collagenase type 2, by Worthington (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Mycoalert, by Lonza (3 mentions)
Apo transferrin, by Merck Group (3 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (3 mentions)
Pronase, by Roche (3 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (3 mentions)
High glucose dmem, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Insulin, by Thermo Fisher Scientific (3 mentions)
72 protocols using ham s f12 media
1
Isolation of Myogenic Mononuclear Cells from Adult Muscle
2
Cell Line Cultivation and Characterization
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Cells were grown in an incubator at 37 °C with 5% CO2. SUM-159 cells received from Dr. Steven P. Ethier (University of Michigan, Ann Arbor, MI) were grown in HAMS F-12 media (ThermoFisher 11765054), supplemented with 6 μg/mL insulin (Sigma I9278), 0.01 M HEPES (Sigma H3375), 1 μg/mL hydrocortisone (Sigma H4001), 1X antibiotic-antimycotic (anti-anti; ThermoFisher 15240062), and 5% FBS (Atlanta Biologicals S11550H). MCF-7 and T47D cells were received from ATCC and grown in DMEM media (ThermoFisher 11965092) containing 1% penicillin/streptomycin (ThermoFisher 15070063) and 10% FBS. Media containing charcoal-stripped bovine serum (Atlanta Biologicals S11650H) was used for indicated assays. For experiments with β-estradiol stimulation, cells were stripped of hormones with media containing CSS for three days prior to stimulation. All cell lines were authenticated by DNA fingerprinting using short tandem repeat (STR) profiling. Cells were routinely tested for mycoplasma using the MycoAlert Mycoplasma Detection kit (Lonza LT07).
3
HPV38-Derived Murine SCC Model
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A UV-induced SCC cell line derived from HPV38E6E7-FVB mice was created in-house (HPV38 SCC cell line) and cultured in modified Ham’s F12 media (Thermo Fisher Scientific, Waltham, MA, USA) as mentioned in previous research [27 (link)]. The HPV38 SCC cell line is a regressor cell line, which is unable to grow in immune-competent animals. HPV38E6E7-FVB mice were thus immunosuppressed by being placed on the TAC-infused diet for 7–8 days prior to subsequent (day 0) with SCC cells on the lower back (subcutaneously s.c. injection, 106 cells/mouse in 100 μL PBS). Mice were maintained on the TAC-infused diet (until removal if relevant as indicated in the figure legends) and tumour growth was recorded every 3–5 days by measuring the major dimension (D) and minor dimension (d) of the tumour via a digital calliper. Measurements were transformed into tumour volume using the formula: tumour volume (cm3) = [D × d2]/2. Euthanasia was performed when the tumour reached a maximum volume of 1 cm3. Histology was performed by the QIMR Berghofer Histology Facility (Brisbane, Australia).
4
Generation of Stable I-A^b-expressing CHO Cell Line
5
Isolation and Culture of HASM Cells
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HASM cells were isolated from deceased, de‐identified lung donors by enzymatic dissociation in accordance with Institutional Review Board approval and as described previously.19 HASM cells were grown in Ham's F‐12 media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, pen/strep, 25 mM HEPES, 1.7 mM CaCl2, and l ‐glutamine. Cells were kept at 5% CO2 and 37°C. Experiments were performed on cells from passage 3‐7 using cells from 10 different donors in total, and at least three different donors for each study. Patient demographics are described in Table 1 . Human fetal lung (HFL‐1) fibroblasts (American Type Culture Collection) were grown in Ham's F12 medium with 10% fetal bovine serum and 1% antibiotic‐antimycotic solution. HEK‐293 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. All cells were kept in a humidified incubator with 5% CO2 at 37°C.
6
TNBC Cell Line Culture and Maintenance
7
Reagents and Materials for Cell Culture
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DMEM low glucose media, DMEM/F-12 media (1:1), Hams’ F-12 media, RPMI 1640 media, penicillin/streptomycin solution, phosphate buffered saline (PBS), Pierce RIPA buffer, and the Presto Blue cell viability reagent were purchased from ThermoFisher Scientific (Waltham, MA). Metformin and the media additives d-biotin, adenine hemisulfate, insulin solution, and apo-transferrin were purchased from Sigma Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Zapoglobin and Isoton II were purchased from Beckman Coulter Inc. (Fullerton, CA). Rabbit anti-mouse IgG secondary antibody was obtained from Zymed Laboratories, Inc. Both horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse antibodies were purchased from GE Healthcare Biosciences (Pittsburg, PA). All tissue culture plasticware and additional chemicals were purchased from ThermoFisher Scientific.
8
Characterization of Cholangiocarcinoma Cell Lines
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Human CCA cells, including KKU-100 (JCRB1568), KKU-213A (JCRB1557), KKU-213C cells [21 (link)], were employed in this study based on our previous report in which these cells showed high FGFR2 protein expression [9 ]. These CCA cells were developed at the Cholangiocarcinoma Research Institute, Khon Kaen University, and deposited in the Japanese Cancer Research Resources Bank (JCRB, Ibaraki city, Osaka, Japan). The CCA cell lines were cultured in Ham's F12 media (21700075, Thermo Fisher Scientific Inc, MA, USA), supplemented with 10 % FBS (10100147, Thermo Fisher Scientific Inc, MA, USA), 10,000 units/mL penicillin-gentamicin, and 10 mM HEPES. All cell lines were incubated at 37 °C in a 5 % CO2 incubator, mycoplasma-negative and used within 10 passages.
9
Culturing TNBC Cell Lines
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Frozen stocks of MDA-MB-231 (RRID:CVCL_0062), CAL-120 (RRID:CVCL_1104), MDA-MB-468 (RRID:CVCL_0419), and CAL-51 (RRID:CVCL_1110) TNBC cells were obtained from ATCC and grown in DMEM (Thermo Fisher Scientific 11965092) supplemented with 10% FBS (Atlanta Biologicals S11650H) and 1% penicillin and/or streptomycin (Thermo Fisher Scientific 15070063). SUM-159 cells (RRID:CVCL_5423) were received courtesy of Steve Ethier and grown in Ham's F-12 media (Thermo Fisher Scientific 11765054) supplemented with 5% FBS, 5 mL of 1 mol/L HEPES (Sigma H3375), 1 μg/mL hydrocortisone (Sigma H4001), 1× antibiotic-antimycotic (Thermo Fisher Scientific 15240062), and 6 μg/mL insulin (Sigma I9278). All cell lines were maintained in a humidified incubator (5% CO2), tested for Mycoplasma monthly (MycoAlert, Lonza LT07), used for no longer than 6 months of continuous culture, and authenticated at the University of Michigan (Ann Arbor, MI) DNA-sequencing core.
10
Isolation and Expansion of Nucleus Pulposus Cells
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NP tissue was obtained from to-be-discarded surgical waste tissues (exempt from IRB review, Washington University Institutional Review Board) of patients (ages 16–75, male and female) receiving surgical treatment for degenerative conditions of the IVD; only demographic information was collected on each patient (age, race, and sex). NP cells were enzymatically isolated from tissues, as previously described (Bridgen et al., 2017 (link); Fearing et al., 2019 (link)). In brief, the NP tissue was digested for 2–4 h at 37 °C [0.4 % collagenase type II (Worthington Biochemical; Lakewood, NJ, USA), 0.2 % pronase (Roche; Basel, Switzerland), and 5 % FBS; 23 mL/g tissue]. Isolated NP cells were passed through a 70 μm filter and then expanded in monolayer culture using Ham’s F12 media (Life Technologies; Carlsbad, CA, USA) supplemented with 1 % penicillin/streptomycin and 10 % FBS under 5 % CO2 and atmospheric O2 at 37 °C. Cells were not used for experimentation past passage 4, and all experiments were conducted using at least 3 human subjects (biological replicates); assay-specific samples sizes are detailed below.
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