We utilized the high grade serous and clear cell carcinoma ovarian cancer cell lines: SKOV3, ATCC (ATCC® #HTB-77™); COV362.4, Sigma Aldrich (Sigma #07071904) and OVCAR4, National Cancer Institute (RRID:CVCL_1627). Cells were maintained in: SKOV3 Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 (DMEM/F12) (Thermo Scientific, #11965118); COV362.4 High glucose Dulbecco’s Modified Eagle Medium (DMEM-HG); and OVCAR4 RPMI-1640 (with sodium bicarbonate; Sigma-Aldrich, #R0883). Media was supplemented with 10% Fetal Calf Serum (FCS) (Thermo Fisher, #16000044) and 1% Penicillin-Streptomycin (Thermo Scientific, #15240062) with all lines maintained at 37 °C with 5% CO2. Cell lines were verified and authenticated by the Australian Genome Research Facility Ltd. Cell counts were conducted prior to experimentation by Trypan Blue viability staining using the Countess® II viable cell count system, cells routinely tested negative for mycoplasma. 3D spheroids were formed by seeding cells into Corning® spheroid Ultra-low adhesion microplates (Sigma-Aldrich #CLS4520) with spheroid integrity, size and shape monitored by live cell microscopy to ensure uniformity across experiments. KRT14 overexpressing and knockout cell lines (LCOE/ LCKO) were generated according to the previously described methods [11 (link)].
Dmem ham s f 12 dmem f 12
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
DMEM/Ham's F-12 (DMEM/F-12) is a cell culture medium used for the in vitro maintenance and growth of a variety of cell types. It is a combination of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cov362, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Eagle s minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Human epidermal growth factor (hegf), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Vibratome, by Thermo Fisher Scientific (1 mentions)
Tissue tek optimum cutting temperature compound, by Avantor (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Ethanol, by Merck Group (1 mentions)
Primocin, by InvivoGen (1 mentions)
Laminin, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
10 protocols using dmem ham s f 12 dmem f 12
1
Ovarian Cancer Cell Culture Protocol
2
Osteoblast and Bone Cancer Cell Culture
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The osteoblasts, hFOB1.19 cells, were cultured in DMEM/Ham’s F-12 (DMEM/F-12; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Sciencell, Carlsbad, CA, USA) and 0.3 mg/mL G418. The MG63 and MNNG/HOS cell lines were grown in Eagle’s minimum essential medium (Thermo Fisher Scientific) containing 10% FBS (Sciencell). The U2OS cells were cultured in RPMI medium 1640 (Thermo Fisher Scientific) supplemented with 10% FBS (Sciencell). Cells were incubated at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.
3
Culturing Human Corneal Epithelial Cells
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Human corneal epithelial (HCE) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HCECs were cultured in Dulbecco's Modified Eagle Medium (DMEM)/Ham's F12 (DMEM/F12, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with hEGF (Peprotech, Rocky Hill, NJ, USA), insulin (Thermo Fisher Scientific), 6% FBS (Thermo Fisher Scientific), and 100 U mL−1 penicillin–streptomycin solution (Thermo Fisher Scientific) in an incubator at 37 °C and 5% CO2. The cells were passaged by 0.25% trypsin containing 0.02% ethylene diamine tetra-acetic acid (EDTA) (Thermo Fisher Scientific) every 3 days.
4
Cell Viability Assay with CCK-8
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Cell viability was examined using the cell counting kit 8 (CCK-8; Dojindo, Kyushu Island, Japan) following the manufacturer’s instructions. Briefly, NPSCs were seeded in 96-well culture plates at a density of 5 × 103 cells per well and treated as above. At indicated time points, the culture medium was discarded, and 100 μL of DMEM/Ham’s F-12 (DMEM/F-12; Thermo Fisher Scientific, Waltham, MA, United States) and 10 μL of CCK-8 reagent were added to each well. Then, the absorbance was detected after incubation for 3 h at 37°C at a wavelength of 450 nm on a microplate reader (Biotek, Winooski, VT, United States).
5
Polymerized High Internal Phase Emulsion Scaffold Fabrication
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Circular disk scaffolds of 15 mm diameter and 200 μm thickness were cut from a cylinder polyHIPE monolith by sectioning using a VT10 0 0 S vibrating-blade microtome (vibratome, Thermo Fisher Scientific). Samples that were too soft to section with a vibratome ( e.g. PEGDA polyHIPE) were embedded in Tissue-Tek® optimum cutting temperature compound (VWR International) in Peel-A-Way® Disposable Histology Moulds (Sigma-Aldrich) and frozen at -20 °C. Cryo-embedded polyHIPE monoliths were then sectioned into 200 μm thick disks using a CM3050-S Cryostat (Leica). Disks were then assembled into Alvetex® Scaffold 12-well inserts (Bio-Scientific Pty Ltd), with the Alvetex® scaffolds removed, and placed in a Falcon® 12-well Clear Flat Bottom Plate (12-well plate, Bio-Strategy), (3.8 cm 2 /well). Scaffolds were sterilised by immersion in 5 mL of 70% w/v ethanol (Merck) in water per well for 30 min under the UV sterilisation light of the biosafety cabinet. Scaffolds were then washed twice with 3.5 mL of 1:1 DMEM/Hams F12 (DMEM/F12), (Thermo Fisher Scientific) per well for 5 min each wash.
6
Immortalized Human Endometrial Stromal Cell Decidualization
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The human ESC-derived telomerase-immortalised cell line, St-T1b (Samalecos et al., 2009 (link)), kindly provided by Professor Jan Brosens (University of Warwick, UK), was maintained in phenol red-free Dulbecco's modified Eagle medium DMEM/Ham's F12 (DMEM/F12; Invitrogen, Renfrew, UK) with 10% steroid-depleted foetal calf serum (FCS) supplemented with 2 mM L-glutamine, 1 μg/ml insulin, 0.3 ng/ml 17β-oestradiol (E2), 50 μg/ml penicillin, 50 μg/ml streptomycin, and 0.2% Primocin (Invivogen, Toulouse, France) (ESC medium) at 37°C in an atmosphere of 5% CO2. Phenol red-free medium was used in all experiments, due to phenol red’s known oestrogenic activity (Berthois et al., 1986 (link)). To induce decidualisation, cells were treated with minimal medium 1 (MM1; ESC medium without insulin and E2) containing increasing concentrations of the progestin, medroxyprogesterone acetate (MPA), 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP; Cambridge Bioscience, Cambridge, UK) and E2, or MM1 with 0.001% ethanol (Table I ), every 48 h, and cultured over 8 days at 37°C in an atmosphere of 5% CO2. All reagents for St-T1b cell culture were purchased from Sigma-Aldrich (Sigma-Aldrich, Dorset, UK) unless stated otherwise. Cultured cells were fixed in 4% paraformaldehyde (PFA) on days 4, 6, and 8 for immunocytochemical analysis.
7
Fibroblast-derived Neural Stem Cells
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The generation and culture of neural stem cells derived from human fibroblasts accomplished by the method developed by Shahbazi and his colleagues [17] . Briefly, human fibroblasts were transduced with lentiviral vectors expressing Zfp521 together with a green fluorescent protein (GFP) tracer (Life Technologies). After viral infection, the cells were cultured in the neural cell culture medium, containing DMEM/Ham's F12 (DMEM/F12; Invitrogen) supplemented with 10% knockout serum replacement (KOSR), 1% non-essential amino acids (NEAA), 1% L-glutamine, ITS (1 mg/ml insulin, 0.55 mg/ml transferrin, 0.67 mg/ml selenium), 1% N2-supplement, 0.05% B27, penicillin/streptomycin (all purchased from Invitrogen), and 1.6 g/l glucose (Sigma-Aldrich). Doxycycline (2 mg/ml) was added to the culture medium to induce Zfp521 expression for 30 days. The epidermal growth factor and basic fibroblast growth factor (EGF, 20 ng/ml and bFGF, 20 ng/ml; Royan Institute) were added to the neural cell culture medium from days 18 to 24. Neurospheres were collected and transferred into a dish and then trypsinized. The isolated cells were subsequently seeded onto tissue culture dishes coated with 0.001% poly-L-ornithine and 1 mg/ml Laminin (Sigma-Aldrich) at 37ºC for 1 hr. and cultured in the neural cell culture medium supplemented with EGF and bFGF in the absence of doxycycline [17] .
8
Isolation and Culture of Fibroblast-Like Synoviocytes
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The collection of synovial samples by needle arthroscopy from RA and OA patients was performed as previously described [23 (link), 24 (link)]. FLS were isolated from the synovial tissues by modified tissue culture method [25 (link)]. Fresh synovial tissues were minced and digested in 1 mg/mL collagenase (Sigma-Aldrich, MO, USA) for 3 h at 37°C to isolate synoviocytes. The cells were cultured with DMEM-Ham's F-12 (DMEM/F12) (Gibco, Shanghai, China), with 20% FCS (Gibco, Mulgrave, VIC, Australia) at 37°C and 5% CO2. FLS from passages three to five were used in this study.
9
Isolation and Culture of Fibroblast-Like Synoviocytes
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RA synovium was collected by closed needle biopsy [26 (link)]. At least six pieces of synovial tissue were obtained per patient to minimize sampling error [27 (link)]. The OA and Orth.A specimens were obtained from knees by closed needle biopsy, arthroplasty or arthroscopy. All samples were fixed in 10% neutral formalin and embedded in paraffin. Sections (5 μm) were cut serially and mounted on adhesive glass slides. Sealed slides were stored at –20°C until staining.
FLS were isolated from the synovial tissues by modified tissue culture method [28 (link)]. Fresh synovial tissues were minced and digested in type I collagenase (Sigma-Aldrich, St Louis, MO, USA). The cells were cultured with DMEM-Ham’s F-12 (DMEM/F12) (Gibco, Life Technologies, Shanghai, China), containing 20% fetal calf serum (Gibco, Life Technologies, Mulgrave, VIC, Australia) in a humidified 5% CO2 incubator. FLS from passages three to five were used in this study.
FLS were isolated from the synovial tissues by modified tissue culture method [28 (link)]. Fresh synovial tissues were minced and digested in type I collagenase (Sigma-Aldrich, St Louis, MO, USA). The cells were cultured with DMEM-Ham’s F-12 (DMEM/F12) (Gibco, Life Technologies, Shanghai, China), containing 20% fetal calf serum (Gibco, Life Technologies, Mulgrave, VIC, Australia) in a humidified 5% CO2 incubator. FLS from passages three to five were used in this study.
10
Cell Culture Protocols for Stem Cell Research
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Human dermal fibroblast cells, STO cells, and PLAT-GP packaging cells were purchased commercially (ATCC, Manassas, VA, USA). The fibroblast growth medium-2 (FGM-2) Bulletkit (Lonza, Tewkesbury, UK) was used for human dermal fibroblast cultures, whereas Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Waltham, MA, USA) containing Glutamax (2 mM; Gibco), penicillin/streptomycin (100 U/mL; Gibco), and fetal bovine serum (FBS, 10%; Life Technologies, Carlsbad, CA, USA) were used for STO and PLAT-GP packaging cells. DMEM/Ham’s F-12 (DMEM/F12; Gibco) medium for human iPSCs and human ES cells contained knockout serum replacement (SR, 20%; Gibco), Glutamax (2 mM; Gibco), nonessential amino acids (NEAA, 0.1 mM; Gibco), 2-mercaptoethanol (0.1 mM; Gibco), 50 U penicillin/streptomycin, and basic fibroblast growth factor (bFGF, 10 μg/mL; Bio-tech/R&D Systems, Minneapolis, MN, USA).
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