BeWo choriocarcinoma cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and were maintained as monolayers in Kaighn's modification of Ham's F-12 medium (Sigma Aldrich, St. Louis, USA) supplemented with 10 % fetal bovine serum (FBS) under standard culture conditions of 5 % CO2 in air at 37 °C with medium renewal every 2–3 day. CYP27B1 knockdown as well as BeWo cell culture supernatants after 1,25(OH)2D3 induction were collected for 25(OH)D and hCG analysis. The shRNA technique was used to knock down the expression of CYP27B1 in BeWo cells. CYP27B1 shRNA target sequence was GGTCAAGGAAGTGCTAAGA. CYP27B1 shRNA plasmid and control plasmid were from Shanghai Qihe Biotechnology Co., Ltd. (China). BeWo were seeded in 6-well culture plates and subsequently infected with CYP27B1 shRNA plasmid or control plasmid at the exponential phase for 48 h, using LipofectamineTm 3000 Transfection Reagent (L3000015, Thermo Fisher, USA). Next, fresh complete medium was substituted for further cultivation. The infection efficiency was evaluated by Western blot 48 h after infection [16 (link)].
Ham s f12 medium
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Switzerland, China
Ham's F12 medium is a cell culture medium developed for the growth and maintenance of various cell types. It is a nutrient-rich formulation that provides the essential components required for cell proliferation and survival. The medium supports the growth of a wide range of cells, including fibroblasts, epithelial cells, and many other cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (24 mentions)
Fetal bovine serum (fbs), by Merck Group (13 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (12 mentions)
Penicillin, by Merck Group (10 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (9 mentions)
Streptomycin, by Merck Group (9 mentions)
Hydrocortisone, by Merck Group (8 mentions)
Dulbecco s modified eagle s medium, by Merck Group (7 mentions)
Rpmi 1640 medium, by Merck Group (6 mentions)
Insulin, by Merck Group (6 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (6 mentions)
L glutamine, by Merck Group (5 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Biowest (5 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
109 protocols using ham s f12 medium
1
Knockdown of CYP27B1 in BeWo Cells
2
Culturing Human Gingival Fibroblasts and Fetal Osteoblasts
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Human Gingival Fibroblasts—HGFs (HGF; Applied Biological Materials Inc., Richmond, BC, Canada) were cultured in a supplemented Dulbecco’s Modified Eagle’s Medium—DMEM (Lonza®, Basel, Switzerland) with 10% Bovine Fetal Serum (Biowest®, Nuaillé, France) and 1% Penicillin with streptomycin (G255 Applied Biological Materials Inc., Richmond, BC, Canada).
Human Fetal Osteoblasts—hFOBs 1.19 (CRL-11372TM; American Culture Collection, Manassas-ATCC®, Manassas, VA, USA) were cultured in a mixture (1:1 v/v) of DMEM (Dulbecco’s modified Eagle’s Medium-DMEM) from (BiowhittakerTM, LonzaTM, Basel, Switzerland) and Ham’s F-12 Medium (Sigma-Aldrich® 51651C, St. Louis, MO, USA), in which was added 0.3 mg·mL−1 of G418 (InvivoGgen, Toulouse, France) and 10% of Bovine Fetal Serum (Biowest®, Nuaillé, France).
Both cell lines were incubated at 37 °C with 5% CO2 and 98% humidity. When the cells reached approximately 80% confluence, trypsin-EDTA (Lonza, Veners, Belgium) was added to detach them, they were later centrifuged, and the pellet was resuspended in the respective medium. To perform each cell culture assay 1 × 104 cells/mL cells at a 4th passage were seeded in 48-well plates containing sterile sample (Corning, NY, USA).
Human Fetal Osteoblasts—hFOBs 1.19 (CRL-11372TM; American Culture Collection, Manassas-ATCC®, Manassas, VA, USA) were cultured in a mixture (1:1 v/v) of DMEM (Dulbecco’s modified Eagle’s Medium-DMEM) from (BiowhittakerTM, LonzaTM, Basel, Switzerland) and Ham’s F-12 Medium (Sigma-Aldrich® 51651C, St. Louis, MO, USA), in which was added 0.3 mg·mL−1 of G418 (InvivoGgen, Toulouse, France) and 10% of Bovine Fetal Serum (Biowest®, Nuaillé, France).
Both cell lines were incubated at 37 °C with 5% CO2 and 98% humidity. When the cells reached approximately 80% confluence, trypsin-EDTA (Lonza, Veners, Belgium) was added to detach them, they were later centrifuged, and the pellet was resuspended in the respective medium. To perform each cell culture assay 1 × 104 cells/mL cells at a 4th passage were seeded in 48-well plates containing sterile sample (Corning, NY, USA).
3
Culturing Bone Cell Lines for Research
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Human normal bone cell line hFOB and osteosarcoma cell lines MG-63 and U2OS were purchased from American Type Culture Collection (ATCC). According to the supplier's protocol, hFOB cells were cultivated in a mixture of 45% Dulbecco's modified Eagle's medium, 45% Ham's F12 Medium and 10% fetal bovine serum (all Sigma-Aldrich; Merck KGaA), U2OS cells were cultured using ATCC-formulated McCoy's 5A Medium (cat no. 30-2007; ATCC) containing 10% fetal bovine serum, whilst MG-63 cells were cultured with Eagle's Minimum Essential Medium (cat no. 30-2003; ATCC) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich; Merck KGaA) maintained in a humidified atmosphere at 37°C under 5% CO2. Cells were harvested upon reaching the logarithmic growth phase for subsequent experiments.
4
Isolation of Stellate Cells from Tissue
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Stellate cells were isolated from the tissues by using of the outgrowth method according to Bachem et al.8 (link). Briefly, the tissue was cut directly after retrieval under sterile conditions into pieces of 3 mm and transferred to culture medium consisting of 400 ml low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (1000 mg/L, Sigma-Aldrich, Germany), 400 ml Ham ´s F12 Medium (Sigma-Aldrich, Germany), 160 ml fetal bovine serum (FBS) (Sigma-Aldrich, Germany), 10 ml penicillin/streptomycin (10.000 Units penicillin and 10 mg streptomycin per ml in 0.9% NaCl, Thermofisher, Germany) and 10 ml amphotericin B (250 μg / ml, Sigma-Aldrich, Germany). The tissues with 5 ml medium were cultured in the incubator at 37 ºC and 5% CO2. Every second day the medium was changed with 5 ml fresh one. The average culture time was three to four weeks until there were enough PSCs for further experiments.
5
HTR-8/SVneo Placental Cell Culture
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HTR-8/SVneo cells derived from human first-trimester placental explant cultures and immortalized by Simian virus (SV40) large T antigen [46 ] were cultured in 1:1 ratio of Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich Inc., St. Louis, MO, USA; #D1152-10L) and Ham’s F-12 medium (Sigma-Aldrich Inc., #N3520-10L) supplemented with 10% fetal bovine serum (FBS; Gibco® Life Technologies, Grand Island, NY, USA; #10270) and antibiotic-antimycotic cocktail [streptomycin (100 units/mL), penicillin (100 µg/mL), and amphotericin B (0.25 µg/mL; MP Biomedicals, Santa Ana, CA, USA; #1674049)] at 37°C under 5% CO2 and 70% relative humidity. Ishikawa endometrial epithelial cells were also cultured as described for HTR-8/SVneo cells. HTR-8/SVneo cells were treated with geneticin sulfate (G418; Himedia, Kelton, PA, USA; #TC025) at 75 μg/mL after every third passage to inhibit the growth of untransfected HTR-8/SVneo cells.
6
Stable COX-2 Overexpression in SUM-149 Cells
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SUM-149 breast cancer cells were obtained from Asterand (Asterand, Inc., Detroit, MI) and maintained in Ham's F12 medium (SIGMA, St. Louis, MO) with 5% calf serum, insulin (5 μg/ml), and hydrocortisone (1 μg/ml). An ~1.8Kb region of the coding sequence of the human COX-2 gene (NM_000963.3) was PCR amplified and cloned into the PCR2.1 Topo vector (Invitrogen, Waltham, MA) and later subcloned between Xho1 and Kpn1 restriction sites in the multiple cloning site (MCS) of a pHAGE-pGK-MCS-Gtx-GFP lentivirus vector. 293T cells (ATCC, Manassas, VA) were co-transfected with the pHAGE-COX-2FL-Gtx-GFP plasmid, the ΔR8.2- packaging plasmid, and a plasmid expressing vesicular stomatitis virus glycoprotein (VSVG) to produce virions. Supernatant containing virions was added to SUM-149 breast cancer cells to derive cells stably expressing the COX-2 gene (SUM-149-COX-2FL). An empty vector without the gene was used to derive control cells (SUM-149-EV). Stable increase of COX-2 expression was verified by PCR and western blot analysis.
7
BeWo Cell Culture Protocol
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BeWo choriocarcinoma cells were cultured in Ham’s F-12 medium (Sigma-Aldrich Inc.) enriched with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies Corp.) supplemented with an antibiotic and antimycotic cocktail consisting of 100 units⁄ml penicillin, 100 μg⁄ml streptomycin and 0.25 μg⁄ml amphotericin B (Biological Industries, Kibbutz beit Haemek). They were maintained at 37 °C in a humidified atmosphere with 5% CO2. The cells were sub-cultured at around 70% confluence.
8
Chondrogenic Mesenchymal Cell Culture
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Ross hybrid chicken embryos of Hamburger–Hamilton stages 22–24 were used to establish primary micromass chondrifying mesenchymal cell cultures. Distal parts of the limb buds of embryos were removed and a single-cell suspension of chondrogenic cells at a density of 1.5×107 cells/mL was yielded. Droplets of different volumes of the cell suspension were inoculated into Petri dishes or plates (Orange Scientifique, Braine-l'Alleud, Belgium). Day of inoculation was considered as day 0. Colonies were fed Ham's F12 medium (Sigma, St. Louis, MO, USA), supplemented with 10% foetal calf serum (Lonza Group Ltd, Basel, Switzerland) and were kept at 37°C in the presence of 5% CO2 and 80% humidity in a CO2 incubator. The medium was changed on every second day. Cultures were maintained for 6 days.
9
HK-2 Cell Culture and Stimulation
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Human renal proximal tubular epithelial cells (HK-2 cells, American Type Culture Collection, Manassas, VA, USA) were cultured, as previously described. Briefly, cells were passaged approximately every three to four days in 100-mm dishes containing combined Dulbecco’s modified Eagle’s (DMEM) and Hams F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Life Technologies; Gaithersburg, MD, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich). The cells were then incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C for 24 h, and sub-cultured until 70–80% confluence. For treatment with recombinant proteins or chemicals, cells were plated onto 60-mm dishes in a medium containing 10% FBS and incubated for 24 h. The cells were then incubated in DMEM-F12 medium with 2% FBS and treated with rhAngII (1 μg/mL; Bachem, Bubendorf, Switzerlandsource) or rhTGFβ (2 ng/mL; R&D Systems) for an additional 16 h. Olmesartan medoxomil (5 ng/mL) was added 1 h prior to rhTGFβ treatment.
10
Cytotoxicity Evaluation of Docetaxel
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RPMI-1640 medium, Ham’s F12 medium, fetal bovine serum (FBS) (mycoplasma-free), penicillin/streptomycin, and trypsin were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Phosphate buffered saline PBS was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Ethylenediaminetetraacetic acid (EDTA), zinc(II) sulphate (BioReagent grade, suitable for cell cultures) and all other chemicals of ACS purity including docetaxel were purchased from Sigma Aldrich Co. (St. Louis, MO, USA) unless noted otherwise.
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