Adenovirus-12 SV40 hybrid transformed immortalized upper airway bronchial epithelial cells (BEAS-2B) were grown in advanced DMEM-F12 media (ThermoFisher, MA) supplemented with 1% FBS, 100 U/mL Penicillin, 100 μg/mL of Streptomycin, 292 μg/mL L-Glutamine and 1% HEPES. hTERT-immortalized human urothelial (TRT-HU1) cell line was grown in DMEM media (ThermoFisher, MA) supplemented with 10% FBS, 100 U/mL Penicillin, 100 μg/mL of Streptomycin and 292 μg/mL L-Glutamine. Cancer cell lines NCI-H520 and HCC15 were authenticated per the Cell lines from the Cancer Cell Line Encyclopedia (CCLE) protocol and grown in RPMI (ThermoFisher, MA) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, MA), 100 U/mL Penicillin, 100 μg/mL of Streptomycin, 292 μg/mL L-Glutamine (Corning, NY). All cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere and tested to ensure the absence of Mycoplasma.
Advanced dmem f12 media
Manufactured by Thermo Fisher Scientific
Sourced in United States
Advanced DMEM-F12 media is a culture medium designed for the growth and maintenance of a variety of cell types, including mammalian and stem cells. It provides a balanced formulation of nutrients, salts, and growth factors to support cell viability and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Corning (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Matrigel, by Corning (3 mentions)
Pensterp cocktail, by Thermo Fisher Scientific (3 mentions)
Ammonium acetate, by MP Biomedicals (3 mentions)
Mgcl2, by MP Biomedicals (3 mentions)
Sodium chloride (nacl), by MP Biomedicals (3 mentions)
Na2hpo4, by MP Biomedicals (3 mentions)
Potassium chloride (kcl), by MP Biomedicals (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ampicillin, by MP Biomedicals (3 mentions)
Kh2po4, by MP Biomedicals (3 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Dmem media, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
22 protocols using advanced dmem f12 media
1
Immortalized Cell Line Maintenance Protocol
2
Culturing Diverse Cell Lines for Research
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Adenovirus-12 SV40 hybrid transformed immortalized upper airway bronchial epithelial cells (BEAS-2B) were grown in advanced DMEM-F12 media (ThermoFisher, MA) supplemented with 1% FBS, 100 U/mL penicillin, 100 μg/mL of streptomycin, 292 μg/mL l -Glutamine and 1% HEPES. hTERT-immortalized human urothelial (TRT-HU1) cell line was grown in DMEM media (ThermoFisher, MA) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL of streptomycin, and 292 μg/mL l -Glutamine. Cancer cell lines NCI-H520 and HCC15 were authenticated per the cell lines from the Cancer Cell Line Encyclopedia (CCLE) protocol and grown in RPMI (ThermoFisher, MA) supplemented with 10% FBS (ThermoFisher, MA), 100 U/mL penicillin, 100 μg/mL of streptomycin, 292 μg/mL l -Glutamine (Corning, NY). All cultures were maintained at 37°C in a humidified 5% CO2 atmosphere and tested to ensure the absence of Mycoplasma.
3
Derivation of Human Intestinal Organoids
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Organoids were derived as previously described5 (link) with some modifications. De-identified human colon and ileum tissue samples from deceased donors were procured by Donor Network West. Tissues were washed with Advanced DMEM/F12 media (ThermoFisher), cut into 5 cm × 5 cm segments, then the epithelium was scraped off from the submucosa into the media and minced with a razor blade. The solution was pelleted at 450 × g for 5 min, then incubated in 2.5 µM EDTA in PBS without Mg2+ or Ca2+ at 37 °C for 9 min (ileum) or 12 min (colon), with vortexing every 3–4 min until crypts were released. Crypts were pelleted at 450 × g for 5 min, washed in PBS, filtered through sterile gauze then a 100 µm cell strainer to remove debris, and pelleted at 450 × g for 5 min. Crypts were resuspended in Cultrex Reduced Growth Factor Basement Membrane Matrix, Type II (BME, R&D Systems cat. no. 3533-010-02) on ice, plated in 50 µL domes in a 24-well plate, cured at 37 °C for 15–30 min, then overlaid with 500 µL Colon Passage Media (Intesticult Organoid Growth Medium (OGM, StemCell Technologies cat. no. 06010) + 10 µM Y-27632) or Ileum Media (OGM + 10 µM Y-27632 + 2.5 µM CHIR99021). After the first 2–3 days in culture, media was changed every 2–3 days, or when media turned yellow, with plain OGM for colon cultures, or Ileum media for ileum cultures.
4
Airway Epithelial Cell Culture Protocol
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Filter-grown airway epithelial cells were measured under open or short circuit conditions as detailed in previous reports [4 (link), 29 (link)]. In brief, PBE cells were grown on Millipore filters in an air-liquid interface (ALI) in Advanced DMEM/F12 media (Thermo Fisher Scientific, USA) supplemented with 0.5 μg/mL hydrocortisone, 100 nM triiodothyronine, and 0.5 μg/mL epinephrine (all from Sigma-Aldrich, Missouri, USA); 0.25 μg/mL human epidermal growth factor (PeproTech, UK); 100 nM TTNPB (Cayman, USA); and 50 nM A83-01 (Tocris Bioscience, Bristol, UK) for 14–21 days. For the first week of ALI, 500 nM A83-01 was supplemented, and for the second week, 10 μM of DAPT (Tocris Bioscience, Bristol, UK) was added.
5
Intestinal Crypt Isolation and Organoid Culture
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Intestinal sections were splayed open, rinsed in PBS, and rotated at 4°C for 45 minutes in Hank’s Buffered Saline Solution with 10 mM EDTA and 1 mM N-Acetyl-L-cysteine (Sigma A9165). Crypts were isolated by scraping with a glass coverslip followed by vortexing and filtering through a 70 uM (small intestine) or 100 uM (colon) cell strainer. Crypt-enriched suspensions were centrifuged at 500xG, 25°C for 5 minutes, washed in PBS, and pelleted again at 500xG, 25°C for 5 minutes. Crypts were plated in Matrigel droplets (Corning 354234) and unless stated otherwise, overlaid with the following “ENR” medium: advanced DMEM/F12 media (Thermo 12634028) containing 1X GlutaMAX (Thermo 35050061), 10 mM HEPES (Thermo 15-630-080), 1X Antibiotic Antimycotic (Thermo 15240062), 1X N-2 Supplement (Thermo 17502048), 1X B-27 Supplement (Thermo 17504044), 5 μM CHIR99021 (Cayman 13122), 1 mM N-Acetyl-L-cysteine (Sigma A9165), 50 ng/mL mEGF (PeproTech 315-09), 5% Noggin/R-spondin conditioned medium (generated using protocol from (32 (link))), and 10 µM Y-27632 (LC Labs Y-5301). Colonic epithelial cultures were fed 50% WRN CM media (57 (link)). Passage-matched enteroids/colonoids were used for all experiments. For induction of CreERT2, enteroids/colonoids were given 1 µM 4-OHT in 100% ethanol at 48 and 24 hours before the start of the time course then mechanically passaged at day 0.
6
Dissociation and Culture of Neuronal Ganglia
7
Cell line authentication and culture conditions
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Cell lines from the Cancer Cell Line Encyclopedia (CCLE) were authenticated per CCLE protocol52 (link) and grown in recommended media supplemented with 10% fetal bovine serum (ThermoFisher, MA) and 100 U/mL Penicillin, 100 μg/mL of Streptomycin, and 292 μg/mL L-Glutamine (Corning, NY). Adenovirus-12 SV40 hybrid transformed bronchial epithelial cells BEAS2B cells were grown in advanced DMEM-F12 media (ThermoFisher, MA) supplemented with 1% fetal bovine serum and 100 U/mL Penicillin, 100 μg/mL of Streptomycin, 292 μg/mL L-Glutamine and 1% HEPES. All cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere and tested to ensure absence of Mycoplasma.
8
Isolation of Testicular Cells from Mice
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Testes from transgenic SCARKO mice and WT litter mates were transferred to 10 ml of digestion buffer 1 (comprised of Advanced DMEM:F12 media [ThermoFisher Scientific], 200 mg/ml Collagenase IV [Sigma], and 400 U/ml DNase I [Sigma]). Tubules were dispersed by gently shaking by hand and allowed to settle for 1 min at room temperature. The supernatant was collected, placed on ice and quenched with the addition of fetal bovine serum (FBS) (Thermo Fisher Scientific). The remaining tubules were then transferred to digestion buffer 2 (0.25 mg/ml trypsin [ThermoFisher Scientific] and 400 U/ml DNase I [Sigma] dissolved in Advanced DMEM:F12 media) and dissociated at 35°C/215 rpm for 5 min each. Any remaining tubule fragments were broken up with manual agitation using a 1000-µl pipette. The resulting single cell suspension was transferred to the previously collected supernatant and quenched with the addition of FBS (Thermo Fisher Scientific). Cells were filtered through a 100-µm strainer, washed in PBS, pelleted at 300 × g for 5 min, and resuspended in MACS buffer containing 0.5% BSA (Miltenyi Biotec). For all Drop-seq experiments, the live single-cell suspensions were collected by flow cytometry using (FACS) ARIA II/III (BD Biosciences) cell sorter.
9
Organoid Culture from Patient-Derived Xenografts
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Organoids were established from PDX tissues as previously described23 (link),44 (link). Fresh or frozen-thawed single PDX cells were seeded in growth factor reduced, phenol red-free, ldEV-free Matrigel (Corning), with 1–1.5 × 105 cells seeded per 20 µl of Matrigel in 48 well plates. Organoids were cultured in organoid media, consisting of advanced DMEM/F-12 media (Thermo Fisher) containing 1% penicillin-streptomycin, 2 mM Glutamax, 1 nM DHT, 1.25 mM N-acetylcysteine, 50 ng/ml EGF, 500 nM A83-01, 10 mM nicotinamide, 10 μM SB202190 (Sigma), 2% B27 (Life Technologies), 100 ng/ml noggin (Peprotech), 10 ng/ml FGF10 (VWR), 5 ng/ml FGF2, 1 μM prostaglandin E2 (Tocris), and 10% R-spondin 1 conditioned media. 10 μM Y-27632 dihydrochloride (Selleck Chemicals) was added to culture medium during organoid establishment and the first subsequent passage. To assess LeY expression, organoids were fixed in 10% formalin for at least 1 h, and then embedded in paraffin before performing immunohistochemistry for LeY, as described above.
10
Ovarian Granulosa Cell Line Culture
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The human ovarian granulosa cell line was kindly gifted by Dr. Zuwei Yang (IPMCH, School of Medicine, Shanghai Jiao Tong University, Shanghai, China). KGN cells firstly were cultured in the glutamine deprived medium (Advanced DMEM/F12 media, Gibco, 12634010) supplemented with 10% fetal calf serum and antibiotics (100 IU/mL penicillin, 100 μg/mL streptomycin) in a 5% CO2 atmosphere at 37 °C for 24 h in order to consume glutamine stored in cells. And then, glutamine and NE were added into the culture medium of KGN cells for another 48 h. NE (HY-13715, MCE, NJ, USA) was solved in dimethyl sulfoxide (DMSO, Yeasen, shanghai, China) at storage concentrations of 100 mM, and treated cells at a concentration of 10 μM diluted with the Advanced DMEM/F12 media. Glutamine (HY-N0390, MCE, NJ, USA) treated cells at a concentration of 2.5 mM or 5 mM diluted with the Advanced DMEM/F12 media.
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