Non-transformed and transformed HBEC3-KT and HBEC3-KTRL53Myc human bronchial epithelial cells were obtained from Dr. John D. Minna, The University of Texas Southwestern Medical Center. NCI-H1573, HCC4019, NCI-H1299, NCI-H2030, NCI-H1703 and NCI-H23 lung cancer cell lines were provided by Dr. Samir Hanash, The University of Texas MD Anderson Cancer Center, whereas 16HBE14o bronchial epithelial cells were provided by Dr. Dieter Gruenert, University of California San Francisco. NCI-H441 lung cancer cells were purchased from the American Type Culture Collection. 16HBE14o cells were cultured in MEM medium (Thermo-Fisher) supplemented with 10% fetal bovine serum (FBS), HEPES, penicillin and streptomycin. H441 cells were cultured in RPMI medium (Thermo-Fisher) supplemented with 5% FBS, dexamethasone (250 μg/ml, Sigma-Aldrich), insulin-transferrin-sodium selenite (Thermo-Fisher), penicillin and streptomycin. H1573, HCC4019, H1299, H2030, H1703 and H23 cells were cultured in RPMI medium supplemented with 10% FBS, HEPES, sodium pyruvate and antibiotics. HBEC3-KT and HBEC3-KTRL53Myc cells were cultured in Keratinocyte serum-free medium (Thermo-Fisher) without antibiotics. The cells were seeded on collagen-coated coverslips for immunolabeling experiments and on 6-well plastic plates for the functional and biochemical studies.
Insulin transferrin sodium selenite
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Insulin-transferrin-sodium selenite is a nutrient supplement for cell culture media. It provides insulin, transferrin, and sodium selenite, which are essential components for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Harvard Bioscience (2 mentions)
Rpmi media, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Tall 104, by American Type Culture Collection (2 mentions)
Recombinant il 2, by Thermo Fisher Scientific (2 mentions)
Mycoprobe mycoplasma detection kit, by R&D Systems (2 mentions)
Tall 104, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Hca 7, by American Type Culture Collection (2 mentions)
Ht 29, by American Type Culture Collection (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
Dmem medium, by Corning (2 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Keratinocyte serum free medium, by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Mycoplasma detection kit, by Minerva Biolabs (1 mentions)
10 protocols using insulin transferrin sodium selenite
1
Culturing Lung Cell Lines for Research
2
Podocyte Culture and Differentiation
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The conditional immortalized human podocytes, control podocytes and podocytes with reduced α-Gal activity were cultured as previously described13 . Briefly, immortalized podocytes were transduced with co-shRNA (control), and with shRNA 894 (α-galactosidase A knockdown) and cultured in RPMI media (Sigma-Aldrich, Taufkirchen, Germany) with insulin-transferrin-sodium selenite as supplement (ThermoFisher Scientific, Waltham, USA) containing 10% fetal bovine serum (Biochrom, Berlin, Germany). Cells proliferated at 33 °C until they reached a confluence of 60–70%. Afterwards cells were differentiated at 37 °C for 14 days before harvesting them for proteomic analysis. Cells were regularly tested for mycoplasma infection using mycoplasma detection kit from Minerva biolabs (Minerva Biolabs, Berlin, Germany).
3
Podocyte Culture and TNFα Stimulation
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Human podocytes60 (link) were cultured in dishes as previously described and regularly tested for mycoplasma using a commercial kit (LookOut, Sigma). 50,000 cells were seeded in 6-well dishes (Thermo Scientific) and cultured at 32 °C with 5% CO2 in RPMI 1640 (Gibco, Thermo Scientific) containing 10% FBS (Gibco, Thermo Scientific), 1% Penicillin-Streptomycin (Gibco, Thermo Scientific), 1% insulin-transferrin-sodium selenite (Thermo Scientific), 1% MEM (Gibco, Thermo Scientific), 1 mM Sodiumpyruvate (Gibco, Thermo Scientific) and 20 mM HEPES (Gibco, Thermo Scientific). 24 h after seeding, cells were washed once with PBS and medium was replaced with FBS-free medium containing 5 ng/mL TNFα (R&D Systems, same vendor as for organoid treatment) or vehicle control. After 24 or 48 h, culture medium was removed and snap frozen. Cells were washed twice with PBS and scraped into ice-cold urea buffer (8 M urea, 100 mM ammonium bicarbonate, 1X PPI), snap frozen and stored until analysis.
4
Immortalized Human Podocyte Culture
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Conditional immortalized human podocytes and podocytes with reduced α-Gal activity were cultured as previously described [11] . Briefly, immortalized podocytes were stably transduced with co-shRNA (control) and shRNA 894 (reducing α-galactosidase A activity to 2-4%, Fig. S1 -for all supplemental material see www.cellphysiolbiochem.com), cultured in RPMI media (Sigma-Aldrich, Taufkirchen, Germany) with insulin-transferrin-sodium selenite as supplement (ThermoFisher Scientific, Waltham, USA) containing 10% fetal bovine serum (Biochrom, Berlin, Germany). Cells proliferated at 33°C until they reached a confluence of 60-70%. Subsequently, cells were differentiated at 37°C for 14 days before harvesting them for further analyses. Cells were regularly tested for mycoplasm infection using the mycoplasm detection kit from Minerva biolabs (Minerva Biolabs, Berlin, Germany).
5
Isolation and Culture of Primary Murine Hepatocytes
6
Propagation and Plaque Purification of SADS-CoV
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IPEC-J2 porcine epithelial cells were propagated in Dulbecco's modified eagle medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Gibco), 1% insulin-transferrin-sodium selenite (Roche), and 5 ng/ml of human epidermal growth factor (Invitrogen). The Vero (ATCC CCL-81) cells were maintained in minimal essential medium (MEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 5% L-glutamine, 100 U/ml of penicillin G, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 incubator (27 (link)). The SADS-CoV CH/FJWT/2018 (passage 10) virus was plaque purified for three times in Vero 81 cells supplemented with a final concentration of 10 μg/ml trypsin in DMEM as described in our previous study (28 (link)).
7
Isolation and Apoptosis Analysis of Intrahepatic BECs
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To isolate the intrahepatic BECs, a two-step liver perfusion was performed. The perfused liver was removed and hepatocytes were selectively removed by forcing them with gentle pressure through an incision in the liver. Cells were suspended in DMEM media supplemented with 10% FBS, 5% NuSerum IV (BD), 0.5 mg/ml insulin–transferrin–sodium selenite (Gibco), 1 mmol/l ascorbic acid 2-phosphate, 10K7 M dexamethasone (Sigma-Aldrich Corp.), 10 ng/ml EGF (R&D, Minneapolis, MN, USA), 10 ng/ml HGF (R&D, Minneapolis, MN, USA). After 10 days cells were exposed to ionomycine 1 μg/ml for 22 hours and percentage of apoptotic cells was determined by flow cytometry using the Annexin V FITC Detection Kit (BD Pharmingen, San Jose, CA).
8
Cell Line Cultivation and Authentication
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HCA-7, HT-29, 293T, and TALL-104 cell lines were obtained from ATCC (Manassas, VA). HCA-7 and HT-29 cells were cultured in McCoy’s 5A medium with 10% FBS. 293T cells were cultured in DMEM medium with 10% FBS. TALL-104 cells were cultured in RPMI1640 medium supplemented with recombinant IL-2 (Peprotech, Cat#200-02) and 20% FBS (Gibco, Cat#10082147). HCEC-1CT cells were cultured in DMEM medium (Corning, Cat#10-013-cv) supplemented with 25 ng/mL EGF (Invitrogen, Cat#PHG0311), 1 μg/mL hydrocortisone (Sigma, Cat#H0888), 1× insulin -transferrin-sodium selenite (Gibco, Cat# 41400045), 50 μg/mL gentamicin (Gibco, Cat#15750-060), and 2% cosmic calf serum (HyClon, Cat# SH30087.02). All cell lines have been tested by MycoProbe Mycoplasma Detection Kit (R&D) and also authenticated before the experiment according ATCC STR database.
9
Cell Line Cultivation and Authentication
10
Isolation and Activation of Murine CD8+ T Cells
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Lymph nodes and spleen were isolated from 6–10-week-old female C57BL6 mice (Jackson Laboratory). CD8a + T cells were purified using the MACs CD8a + T cell isolation kit (Miltenyi) according to manufacturer’s instructions. Cells were plated (5 × 106 cells/well) in 6 well dishes coated with 2.5 µg/mL anti-CD3 (BioLegend) and 5 µg/mL anti-CD28 (BD Pharmigen) in RPMI 1640 + GlutaMAX (Gibco) supplemented with 10% HD FBS (Gibco), 1% insulin-transferrin-sodium selenite (Gibco), 50 µM 2-mercaptoethanol (National Diagnostics), 1X penicillin, and 1X streptomycin (Caisson Labs). On day 1, media was supplemented with 0.5 ng/mL IL-7 (BioLegend) and 20 ng/mL IL-2 (BioLegend). On day 2, cells were split (1 × 106 cells/well) into uncoated 6 well dishes. On day 4, cells were harvested for use and subjected to described experimental conditions.
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