Fibroblasts were cultured to confluence (~30,000 cells/well) in 96-well white plates (Greiner Bio-One, Gloucestershire, UK) using ADMEM (ThermoScientific) containing 10% FBS and 1% penicillin–streptomycin. Cells were synchronized with 200 nM dexamethasone, diluted in serum-free medium for an hour and washed twice with the same solution (ThermoScientific) before reconstituting with serum-free medium containing 1× B27 (ThermoScientific) and 400 μM luciferin (Gold Biotechnology, St. Louis, Missouri, USA). Bioluminescence was recorded for 4 days in Tecan M200 Pro readers. Actimetrics MultiCycle was used to determine the period and amplitude (baseline subtracted 24 h running average of the raw luminescence data smoothed over 8 h). For luminometric experiments involving the 62 fibroblast cell lines, three technical replicates per dose, per drug, were conducted once and averaged.
Admem
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
ADMEM is a basal cell culture medium formulated for the growth and maintenance of adherent cell lines. It provides the basic nutritional requirements for cell growth and supports a variety of mammalian cell types.
Automatically generated - may contain errors
Lab products found in correlation
Luciferin, by GoldBio (2 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (2 mentions)
White 96 well plate, by Greiner (2 mentions)
M200 pro readers, by Tecan (2 mentions)
Multicycle, by Actimetrics (2 mentions)
Neomycin psn antibiotic, by Thermo Fisher Scientific (1 mentions)
Atcc crl 1586, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ham s f12 medium, by Merck Group (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
6 protocols using admem
1
Circadian Rhythm Assay in Fibroblasts
2
Dermal Fibroblast Isolation and Culture
3
Circadian Rhythm Profiling in Fibroblasts
4
Dermal Fibroblast Culture Methodology
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BD patient-derived dermal fibroblasts (n = 39) and control subject-derived dermal fibroblasts (n = 23) were obtained from the StemBANCC consortia, who oversaw the collection and culturing of cells from 500 individuals under standardized conditions. Fibroblasts were derived from 3 mm punch-skin biopsies, following informed consent in accordance with StemBANCC ethics practice [36 ]. Biopsies were plated in Advanced DMEM (ADMEM, ThermoFisher Scientific, Waltham, Massachusetts, USA) with 10% Fetal Bovine Serum (FBS) for outgrowth of fibroblasts, with subsequent expansion for generating a banked frozen stock in 10% DMSO at passage 2 in nitrogen vapor. This was further expanded for functional studies, which were carried out at passages 4–6.
5
Vero E6 Cell Culture for SARS-CoV-2 Isolation
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Vero E6 cells were used for SARS‐CoV‐2 isolation attempts. The cells had been obtained from the American Type Culture Collection (catalog no. ATCC CRL‐1586) and have been used for our SARS‐CoV‐2 projects,30 , 31 , 32 , 33 including the isolation of >30 SARS‐CoV‐2 isolates from human and environmental samples. The cells were propagated in a cell culture medium comprised of advanced Dulbecco's modified essential medium (aDMEM; Invitrogen) supplemented with 10% low antibody, heat‐inactivated, gamma‐irradiated fetal bovine serum (FBS; Hyclone, GE Healthcare Life Sciences), l ‐alanine, l ‐glutamine dipeptide supplement (GlutaMAX,), and 50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin (PSN antibiotics, Invitrogen) with incubation at 37°C in 5% CO2.
6
Urothelial Cancer Cell Culture and Lipid Extraction
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The RT4 and T24 urothelial cancer cell cultures were maintained as in Imani et al. (2012). 24 Briefly, both of these human urothelial This journal is © The Royal Society of Chemistry 2016 cancer cell lines were cultured in a 1 : 1 mixture of Advanced-Dulbecco's Modified Essential Medium (ADMEM, Invitrogen, Gibco, Paisley, UK) and Ham's F-12 medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 5% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA), 100 mg ml À1 streptomycin and 100 units per ml penicillin. The RT4 and T24 urothelial cancer cell cultures were plated simultaneously with a seeding density of 5 Â 10 4 cells per cm 2 . To achieve sufficient biomass for NMR analysis, two flasks (2 Â 75 cm 2 ) of RT4 cultures and three flasks (3 Â 75 cm 2 ) of T24 cultures were necessary (approximately 3 Â 10 7 cells in each case). The cells were incubated at 37 1C in a humidified atmosphere of 5% CO2 for 1 week to achieve confluence. The cells were then detached with TrypLE Select (Gibco, Invitrogen), resuspended in the cell growth medium, and centrifuged at 200 Â g for 5 min. The cell pellets were collected for lipid extraction and analysis. RT4 and T24 cells were both grown repeatedly in 3 different months.
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