Immortalized Beas2B cells, and NSCLC cells, H460, A549, H838, H520, H522, H2009, H2030 and murine Lewis lung cancer (LLC) were obtained from the ATCC. Human bronchial epithelial cells (HBECs) immortalized with CDK4 and hTERT were provided by John Minna, MD, PhD (UT Southwestern). HBECs were cultured with keratinocyte serum-free medium (KSFM; Life Technologies Inc.) media containing 50 μg/mL of bovine pituitary extract (BPE; Life Technologies Inc.) and 5 ng/mL of EGF (Life Technologies Inc.). Beas2B cells were grown in keratinocyte serum free media with 5 pg/mL human EGF and 0.05 mg/mL bovine pituitary extract (Gibco, Invitrogen). NSCLC cells were grown in RPMI 1640 supplemented with 10% calf serum (Biofluids) (R10). H838 cells were grown with R10 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate. LLC cells were grown with DMEM media supplemented with 10% calf serum. The authenticity of these cell lines were verified by short tandem repeat analysis (Johns Hopkins). Rapamycin (Calbiochem) and 4EGI-1 (Chembridge Corp., San Diego, CA) were dissolved in 100% DMSO, aliquoted, and stored at -20°C until used. Gemcitabine (Eli Lilly and Co.) was dissolved in 1X PBS and stored at room temperature.
Bovine pituitary extract
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Canada, Germany, Italy, Japan, Denmark
Bovine pituitary extract is a cell culture supplement derived from the pituitary glands of bovine (cattle) origin. It is commonly used as a growth-promoting additive in cell culture media to support the proliferation and survival of various cell types.
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Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (147 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (120 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (85 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (66 mentions)
Penicillin, by Thermo Fisher Scientific (48 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (45 mentions)
Streptomycin, by Thermo Fisher Scientific (42 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (40 mentions)
Lncap, by American Type Culture Collection (32 mentions)
Hydrocortisone, by Merck Group (30 mentions)
Du145, by American Type Culture Collection (29 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (28 mentions)
Keratinocyte sfm, by Thermo Fisher Scientific (28 mentions)
Rwpe 1, by American Type Culture Collection (28 mentions)
Recombinant epidermal growth factor, by Thermo Fisher Scientific (26 mentions)
Rwpe 1, by Thermo Fisher Scientific (26 mentions)
L glutamine, by Thermo Fisher Scientific (24 mentions)
Rpmi 1640, by Thermo Fisher Scientific (22 mentions)
Insulin, by Merck Group (21 mentions)
Cholera toxin, by Merck Group (20 mentions)
529 protocols using bovine pituitary extract
1
Cell Line Characterization and Treatment
2
Immortalized Vaginal Epithelial Cell Line
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The WT VK2/E6E7 vaginal epithelial cell line was obtained from the ATCC (Manassas, VA, USA). This cell line (ATCC CRL-2616) was derived from normal human vaginal mucosal tissue and immortalized at passage 3 by transduction with the retroviral vector LXSN-16E6E7 in the presence of polybrene. The VK2 cells and the two TRIM26-modified VK2 variants (see below) were grown and maintained in keratinocyte serum-free medium (KSFM). KSFM was supplemented with an additional 0.1 ng/mL of human recombinant epidermal growth factor, 0.05 mg/mL of bovine pituitary extract, 0.4 mM CaCl2, and 100 units/mL penicillin-streptomycin (Life Technologies, Carlsbad, CA, US), as described before [21 (link)]. KSFM, human recombinant epidermal growth factor, and bovine pituitary extract were obtained from Life Technologies. Human embryonic kidney (HEK) 293T cells containing the SV40 T-antigen (ATCC Manassas) were cultured in high glucose formulation of Dulbecco’s modified eagle’s medium (DMEM) with 4.5 g/L of glucose and supplemented with 10% fetal bovine serum (FBS). African green monkey kidney or Vero cells (ATCC) were cultured in minimum essential medium eagle-alpha modification (α-MEM) supplemented with 5% of FBS and 1% of l -glutamine, penicillin-streptomycin, and HEPES.
3
Organotypic Skin Cultures with Dermal Fibroblasts
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Primary human keratinocytes were cultured in Keratinocyte Serum Free Medium supplemented with Epidermal Growth Factor 1–53 and Bovine Pituitary Extract (Life Technologies; 17005042). Generation of organotypic skin cultures were performed as described in Li and Sen, 2015. Briefly, ~500K control cells were seeded on devitalized human dermis and raised to an air/liquid interface to induce differentiation and stratification over the indicated number of days with culture changes every two days. Prior to seeding keratinocytes, either Matrigel was applied to the underside of the devitalized dermis or primary human dermal fibroblasts were centrifuged into the devitalized dermis. To evaluate the effect of oxygen levels on 3D skin cultures, FibHSEs were cultured as previously described and exposed to either normoxia (18–20% oxygen) or hypoxia (3% oxygen) at the air-liquid interface for 14 days. To measure changes from EGF supplementation, culture medium was switched to Keratinocyte Serum Free Medium supplemented with Bovine Pituitary Extract and variable concentrations of Epidermal Growth Factor 1–53 (Life Technologies; 17005042) after one week for one additional week of culturing.
4
Organotypic Skin Cultures with Dermal Fibroblasts
5
Cell Culture Protocols for NHF and SCC13 Cells
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NHF were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L glucose, stable glutamine, sodium pyruvate and 3.7 g/L NaHCO3 (PAN Biotech), supplemented with 10% foetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (PAN Biotech). SCC13 cells were maintained in Keratinocyte Serum Free Medium (SFM) with l -Glutamine (Gibco), supplemented with 2.5 μg epidermal growth factor and 25 mg bovine pituitary extract (Gibco), as well as 1% penicillin/streptomycin. All cells were kept at 37 °C with 5% CO2. To passage at confluency, cells were washed with 1× phosphate-buffered saline solution (1×-PBS, PAN Biotech) and detached with 0.05% trypsin/0.02% EDTA in PBS without Ca and Mg (PAN Biotech).
6
Culture and Maintenance of Esophageal Cell Lines
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Human esophageal epithelial cells, STR, were cultured as previously described. In brief, Keratinocyte-SFM medium was supplemented with 1 ng/mL Epidermal Growth Factor, 0.05 mg/mL Bovine Pituitary Extract, and 1% penicillin streptomycin antibiotics (Gibco™, for Life Technologies, Inc., Carlsbad, CA, USA). Bile/acid-tolerant STR cells were established by repeated exposure to a 100 μM of cocktail of bile salts (20 μM of each, deoxycholic acid (MP Biomedicals, Santa Ana, CA, USA), glycocholic acid, taurocholic acid, sodium glycodeoxycholate and sodium glycochenodeoxycholate (all Sigma, St. Louis, MO, USA) as reported) [34 (link)].The non-neoplastic Barrett’s epithelial cell lines CPA, CPB, BAR-T and BAR-10T cells were isolated from non-dysplastic metaplasia (kind gift from Dr. Rhonda Souza, Baylor Scott White) [35 (link)], and were cultured in Epithelial Cell Medium-2 supplemented with 5% epithelial cell growth supplement-2 (EpiCGS-2) and 5% penicillin/streptomycin antibiotics (ScienceCell™ Research Laboratories, Carlsbad, CA, USA). The esophageal adenocarcinoma cell lines, FLO1, OE33 and OE19 (kind gift of Dr. El-Rifai, University of Miami), were grown in RPMI with 10% FBS and 1% penicillin/streptomycin antibiotics (ThermoFisher Scientific, Waltham, MA, USA). All cells were incubated at 37 °C with 5% CO2.
7
Characterization of Prostate Cell Lines
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Five cell lines provided by Linkou Chang Gung Memorial Hospital were examined in this study, including four PCa cancer cell lines (22RV1, DU-145, LNCaP, and PC-3) and one immortalized normal prostate cell (PZ-HPV-7). The cell lines 22RV1, DU-145, and PC-3 contain a TP53 gene missense mutation; the LNCaP cell carries a gene AR missense mutation. All PCa cells were cultured in Roswell Park Memorial Institute 1640 Medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). PZ-HPV-7 was cultured in serum-free medium Keratinocyte-SFM (Gibco, Grand Island, NY, USA) mixed with 20–30 μg/mL bovine pituitary extract (Gibco, Grand Island, NY, USA) and 0.1–0.2 ng/mL recombinant EGF (Gibco, Grand Island, NY, USA). Quantities of 1 mL/500 mL MycoZapTM Plus-PR (LONZA, VZA-2021, Basel, Switzerland) antibiotics were added to each basal medium at 37 °C in a humidified atmosphere of 5% CO2. After harvesting, the cells were extracted for further mRNA, microRNA, protein, and cell viability analyses.
8
Dose-Dependent Effects of Diatrizoate on HK-2 Cells
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Non-cancerous human immortalized kidney epithelial cells (HK-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA, Item No. CRL-2190) and were cultured according to ATCC guidelines. Cells were grown in keratinocyte-free media with added bovine pituitary extract (50 μg/mL) and recombinant epithelial growth factor (5 ng/mL) purchased from Fisher Scientific (Gibco, Carlsbad, CA, USA, Item No. 17005-042). Cells were grown in a warm, humidified incubator set to 37 °C with 5% CO2. HK-2 cells were plated into six-well cell culture plates (Corning, Sigma Aldrich Item No. CLS3516) at a cellular density of 750,000 cells/mL and allowed to grow for 48 h prior. Media was subsequently replaced and cells were treated with a final concentration of 0, 2, 5, 10, 15, 18, 23, 28, or 30 mg I/mL DA for 2, 8, or 24 h prior to cell lysate collection. Vehicle control was an equal volume of PBS. Following the treatment period, media was removed via aspiration and cells were collected using trypsin–EDTA (0.25%) (Gibco, Carlsbad, CA, USA, Item No. 25200-072) for sample analysis.
9
Culturing Oral Keratinocytes and KSHV Production
10
Culturing Bronchial Epithelial Cell Lines
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The human bronchial epithelial cell line BEAS-2B was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in serum-free Bronchial Epithelial Cell Growth Medium (BEGM) medium (Lonza, Walkersville, MD, USA), supplemented with Bronchial Epithelial Cell Growth Medium SingleQuots™ Supplements and Growth Factors (Lonza). The HBEC-3KT bronchial epithelial cell line was kindly provided by Professor Jerry Shay (UT Southwestern, TX, USA). The cells were immortalized in the laboratory of Prof. Shay and further cultured as described by Ramirez et al. [29 (link)]. In brief, the cells were cultured in KSF (Keratinocyte Serum-Free) media (Gibco, Carlsbad, CA, USA) supplemented with epidermal growth factor 1-53 (EGF 1-53) and Bovine Pituitary Extract (BPE) provided frozen from the manufacturer. The MRC-9 normal human lung fibroblasts were purchased from the ATTC. The cells were expanded and maintained in Eagle's Minimum Essential Medium (Sigma-Aldrich, St. Louise, MO, USA) supplemented with fetal bovine serum and L-glutamine as instructed by the supplier.
The cells were stimulated with 0.1-0.2 ng/ml recombinant human TGF-β1 (#240-B-002, R&D Systems, Minneapolis, USA).
The cells were stimulated with 0.1-0.2 ng/ml recombinant human TGF-β1 (#240-B-002, R&D Systems, Minneapolis, USA).
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