Patient and control fibroblasts were cultured in Dulbecco's modified Eagle's medium (high glucose; Life Technologies, Karlsruhe, Germany) containing 10% foetal calf serum (PAN Biotech, Aidenbach, Germany) and 1% Pen/Strep at 5% CO2 at 37°C. Growth medium was changed every 72 h. For the sugar supplementation studies, L‐fucose (100 µM in PBS) was added to the growth medium for 48 h. For viral infection of fibroblasts, the ecotropic packaging cell line FNX‐Eco (ATCC) and the amphotropic packaging cell line RetroPack PT67 (Clontech) were cultured in DMEM containing 1 × L‐glutamine, 1× Pen/Strep and 10% foetal calf serum (PAN Biotech GmbH; heat‐inactivated at 65°C for 30 min) at 37°C under 5% CO2.
Foetal calf serum
Manufactured by PAN Biotech
Sourced in Germany, France
Foetal calf serum is a complex mixture of proteins, hormones, and other growth factors derived from the blood of unborn calves. It is commonly used as a nutritional supplement in cell culture media to support the growth and proliferation of a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by PAN Biotech (3 mentions)
Dmem high glucose, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Hepg2, by American Type Culture Collection (2 mentions)
L glutamine, by PAN Biotech (2 mentions)
High glucose dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Pen strep, by PAN Biotech (1 mentions)
Dmem f12, by PAN Biotech (1 mentions)
Dulbecco modified eagle medium (dmem), by PAN Biotech (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by PAN Biotech (1 mentions)
Dmem f12 medium, by PAN Biotech (1 mentions)
Mcf 7, by PAN Biotech (1 mentions)
α mem medium, by Merck Group (1 mentions)
Rpmi medium, by PAN Biotech (1 mentions)
Hek293 cells, by Leibniz Institute DSMZ (1 mentions)
Dulbecco s modified eagles s medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using foetal calf serum
1
Fibroblast Culture and Viral Infection
2
Cell Culture Conditions and Derivation
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All cells were cultured in a humidified incubator at 37 °C, 5% CO2 in DMEM10% (unless otherwise indicated). DMEM10% consists of DMEM high-glucose (Sigma-Aldrich) supplemented with 10% foetal calf serum (PanBiotech), 100 mg/l streptomycin (Sigma-Aldrich) and 60 mg/l penicillin (Sigma-Aldrich). Production of stable cell lines was done as described before [24 (link)]. MEFs double deficient for iRhom1 and iRhom2 were obtained from indicated mouse lines as described before [19 (link), 24 (link)]. HEK293 cells were purchased from the German Collection of Microorganisms and Cell Cultures (GmbH DSMZ- No. ACC 305). Normal human epidermal keratinocytes and human dermal fibroblasts were isolated from human skin samples as described before [94 (link)]. Primary keratinocytes were cultivated in Dermalife K—Life Line (Cell Systems LL-0007). Primary fibroblasts were cultivated in DMEM10%.
3
Cancer Cell Line Culturing Protocol
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The breast cancer cell lines MCF-7 and MDA MB-231 were a kind gift from Göran Landberg (Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden). The colon cancer cell line HT-29, the hepatocellular cancer cell line HEPG2, the cervical cancer cell line HeLa and the adenocarcinoma lung cancer cell lines A549 and H441, as well as the non-cancerous cell line HEK-293, were purchased from the American Type Culture Collection (ATCC). The glioblastoma cell line U251 was a kind gift from Kai Murk (Charité Berlin, Germany). A549, HEK-293, HeLa, HEPG2, HT-29, MCF-7 and U251 cells were cultured in DMEM, and H441 and MDA-MB-231 cells were cultured in DMEM/F12. All media contained penicillin/streptomycin (100 U ml− 1), L-glutamine (DMEM: 584 mg l− 1, DMEM/F12: 365.1 mg l− 1) and 10% heat-inactivated foetal calf serum (PAN Biotech, Germany). The humidified incubator was set at 37 °C with 5% CO2. Cells were harvested using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) in PBS.
4
Culturing and Characterizing Human Cell Lines
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The following human cell lines were used: colorectal, T84; lung, NCI-H358; ovarian, SKOV3; gastric, AGS; esophageal, OE33; hepatoma, Huh7 and breast, MCF7; all of them being obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). T84 were cultured in DMEM-F12 medium (PAN-Biotech). SKOV3, OE33, NCI-H358 were cultured in RPMI medium (PAN-Biotech). MCF-7 were cultivated in MEM Eagle with EBSS medium (PAN-Biotech). AGS were cultivated in F12-K medium (Mediatech, Inc.) and Huh7 were cultivated in α-MEM medium (Sigma-Aldrich) supplemented with non-essential amino acids (Gibco) and sodium pyruvate (PAN-Biotech). All culture medium were supplemented with 10% foetal calf serum (PAN-Biotech) and 1% v/v penicillin/streptomycin (PAN-Biotech). Absence of mycoplasma infection was confirmed by regular testing.
5
Culturing HEK293 and MEF iRhom1/2 KO Cells
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Culturing of HEK293 cells and MEFs, double deficient for iRhom1 and iRhom2 (MEF_dKO): in a humidified incubator at 37 °C, 5% CO2 in DMEM10%, which consists of DMEM high-glucose (Sigma-Aldrich) supplemented with 10% foetal calf serum (PanBiotech), 100 mg/l streptomycin (Sigma-Aldrich) and 60 mg/l penicillin (Sigma-Aldrich). Generating of stable cells was performed as described before [28 (link)]. MEFs were obtained from indicated mouse lines as described before [19 (link)]. BMDMs were freshly isolated from femur and tibiae of respective mouse lines as described previously [42 (link)]. HEK293 cells were purchased from German Collection of Microorganisms and Cell Cultures (GmbH DSMZ- No. ACC 305).
6
HNSCC Cell Line Cultivation
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CAL27 and CAL33 HNSCC cell lines13 (link) were kindly provided by Dr J.L. Fischel (Centre Antoine Lacassagne, Nice, France, where they were initially obtained). They were authenticated by Azur Génétique (Nice, France) as identical to the corresponding DSMZ cell lines (report reference AGLC-14-00225). They were cultured in Dulbecco’s modified Eagles’s medium (Invitrogen, Cergy-Pontoise, France) supplemented with 10% heat-inactivated foetal calf serum (Pan Biotech) and maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Petri dishes or flasks of various sizes. They were replicated every 4–5 days, and the medium was changed once in-between.
7
Preparation of AR42J Cell Membranes
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The SST2-expressing AR42J, acinar pancreatic tumour cell line derived from a transplantable tumour of a rat exocrine pancreas, was provided by ATCC (LGC Standards, Molsheim, France). Cells were cultured in RPMI 1640 medium (Dutscher, Bernolsheim, France), supplemented with 10% foetal calf serum (PAN-Biotech, Aidenbach, Germany), in a humidified atmosphere at 37 °C with 5% CO2. AR42J membranes were obtained by sonication in 50 mM Tris–HCl, pH 7.4, and centrifugation at 39,000× g for 10 min at 4 °C. The pellet was resuspended in the same buffer and centrifuged at 50,000 ×g for 10 min at 4 °C, and membranes in the resulting pellet were stored at −80 °C until further use.
8
Cell Culture Protocols for Cancer Research
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The breast cancer cell lines MCF-7 and MDA MB-231 were a kind gift from Göran Landberg (Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden). The colon cancer cell line HT-29, the hepatocellular cancer cell line HEPG2, the cervical cancer cell line HeLa and the adenocarcinoma lung cancer cell lines A549 and H441, as well as the non-cancerous cell line HEK-293, were purchased from the American Type Culture Collection (ATCC). The glioblastoma cell line U251 was a kind gift from Kai Murk (Charité Berlin, Germany). A549, HEK-293, HeLa, HEPG2, HT-29, MCF-7 and U251 cells were cultured in DMEM and H441 and MDA-MB-231 cells in DMEM/F12. All media contained penicillin/streptomycin (100 U ml - 1 ), L-glutamine (DMEM: 584 mg l - 1 , DMEM/F12: 365.1 mg l - 1 ) and 10% heat-inactivated foetal calf serum (PAN Biotech, Germany). The humidi ed incubator was set at 37 °C with 5% CO 2 . Cells were harvested using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) in PBS.
9
Calu-3 Cell Culture for Airway Research
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Calu-3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA).
The cells were cultured in DMEM/Ham's F12 (1/1) supplemented with L-glutamine (2 mM) and 10% foetal calf serum (PAN-Biotech GmbH, Aidenbach, Germany) and incubated at 37°C under 90-95% of relative humidity and 5% v/v of CO 2 in air. The Calu-3 cells at passages 50-60 were seeded at a density of 15 x 10 4 cells/cm 2 onto 12-well plate Transwell inserts (Corning Transwell Clear PET membrane 0.4µm, Thermofischer Scientific). The cells were cultured under air-interface conditions for 15 days. The growth medium in the basolateral compartment (1.5 ml) was replaced by fresh medium every other day.
The cells were cultured in DMEM/Ham's F12 (1/1) supplemented with L-glutamine (2 mM) and 10% foetal calf serum (PAN-Biotech GmbH, Aidenbach, Germany) and incubated at 37°C under 90-95% of relative humidity and 5% v/v of CO 2 in air. The Calu-3 cells at passages 50-60 were seeded at a density of 15 x 10 4 cells/cm 2 onto 12-well plate Transwell inserts (Corning Transwell Clear PET membrane 0.4µm, Thermofischer Scientific). The cells were cultured under air-interface conditions for 15 days. The growth medium in the basolateral compartment (1.5 ml) was replaced by fresh medium every other day.
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