A total of 5 × 104 WT or DOXR cells were seeded in 35 mm culture dishes (Falcon, USA) and maintained in growth medium. Both cell types were collected using trypsin and counted using a Luna automated cell counter (Logos Biosystems, Korea) after 24, 48, and 72 h.
35 mm culture dishes
Manufactured by BD
Sourced in United States
35-mm culture dishes are laboratory equipment used for the cultivation and maintenance of cells in a controlled environment. These dishes provide a standardized and sterile container for cell culture experiments.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dnase, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Dnase, by Thermo Fisher Scientific (2 mentions)
Laminin, by Merck Group (2 mentions)
Luna automated cell counter, by Logos Biosystems (1 mentions)
Glass bottom dishes, by Iwaki (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by BD (1 mentions)
Poly l lysine solution, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Mem neaa 100, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
24 well plate, by Techno Plastic Products (1 mentions)
Puromycin, by Merck Group (1 mentions)
15 protocols using 35 mm culture dishes
1
Cell Growth Kinetics Monitoring
2
CHO Cell Culture Protocol for Microscopy
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CHO cells (a kind gift from Dr Yu at Keio University, originally obtained from American type culture collection and not tested for mycoplasma infection) were maintained in culture media (10% fetal bovine serum in Dulbecco's modified Eagle's medium supplemented with penicillin and streptomycin) in a humidified 5% CO2 incubator at 37 °C until use. For imaging, CHO cells were plated on poly-L -lysine (100 μg ml−1 in 0.1 M borate buffer, pH 8.5)-coated coverslips (Fisher Scientific, ϕ12 mm, ∼0.15 mm thickness) in 35-mm culture dishes (Falcon) or glass-bottom dishes (Iwaki, ϕ12 mm, ∼0.15 mm thickness) coated in the same manner at a density of 5 × 104 cells per 35-mm dish.
3
Cell Culture of A431 and HO-1-N-1 Carcinoma Lines
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We used the epidermoid (squamous cell) carcinoma cell line A431 (RRID:CVCL_0037) and OSCC cell line HO-1-N-1 (RRID:CVCL_1284). Both cell lines were cultured in serum-free medium, as previously described [18 (link),19 (link),20 (link)]. Briefly, the cells were routinely grown in 35 mm culture dishes (BD Falcon, San Jose, CA, USA) in DF6F medium composed of a 1:1 mixture, by volume, of Dulbecco’s modified Eagle medium and Ham F-12 medium supplemented with 10 μg/mL of insulin, 5 μg/mL of transferrin, 10 μM of 2-aminoethanol, 10 nM of sodium selenite, 10 μM of 2-mercaptoethanol, and 9.4 μg/mL of oleic acid conjugated with fatty acid-free bovine serum albumin. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents used for cell culture were free of mycoplasma and viral pathogens. A431 and HO-1-N-1 cells were cultured at 37 °C in a humidified 95% air/5% CO2 atmosphere in a CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) until they were grown to 60–70% confluency.
These cell lines are free from mycoplasma contamination and have been authenticated using short tandem repeat profiling (BEX CO., LTD. Tokyo, Japan), and the profiles of A431 and HO-1-N-1 matched with the publicly available reference profiles, as mentioned previously [15 (link),16 (link)].
These cell lines are free from mycoplasma contamination and have been authenticated using short tandem repeat profiling (BEX CO., LTD. Tokyo, Japan), and the profiles of A431 and HO-1-N-1 matched with the publicly available reference profiles, as mentioned previously [15 (link),16 (link)].
4
Zebrafish Swim Bladder Inflation
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Zebrafish larvae of the WT and cyp20a1-/- mutant line were kept until 6 dpf in 35 mm culture dishes (Falcon) containing 10 larvae in 10 ml per dish. At 6 dpf, the larvae were visually compared using a stereomicroscope, scored based on swim bladder inflation, and imaged. This experiment was independently repeated three times with six dishes per line (WT, wh61) and experiment (total n = 18). The morphological appearances of adult WT and mutant fish were also compared during the novel tank assay.
5
Culturing Postnatal Suprachiasmatic Nucleus Neurons
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Postnatal day 2–5 newborn rats were euthanized by decapitation. SCN regions were dissected from ~600 μm thick hypothalamic slices and cells were dissociated after treatment with trypsin, according to published methods (Watanabe et al., 1993 (link); Bhattacharya et al., 2013 (link)). Next, cells were purified on a discontinuous protein gradient, and ~100,000 cells were placed on coverslips coated with a 1% poly-L-lysine solution (Sigma) in 35 mm culture dishes (BD Falcon) and cultured in Neurobasal A medium with 2% B27 supplement and 0.5 mM L-Glutamine in a humidified CO2-containing atmosphere at 37°C until use (14–21 days).
6
HeLa Cell Culture and Transfection
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HeLa CCL-2 (ATCC) cells were grown in DMEM medium (Life Technologies) with 10% FBS (Life Technologies), 0.5 mM L-glutamine (Life Technologies), 1% MEM NEAA 100× (Life Technologies), and 1% penicillin/streptomycin (10,000 U/ml; Life Technologies) at 37°C and 5% CO2. HeLa cells were transfected using FuGENE 6 transfection reagent (Promega) according to the manufacturer’s instructions. For cryo-ET, ∼25,000 cells were seeded in 35-mm culture dishes (BD Falcon) containing four pre-treated EM grids 24 h before transfection. For Western blot experiments, cells were plated in 6 cm dishes 24 h before transfection (250,000 cells/well). To inhibit autophagy, cells were treated with 50 μM chloroquine in water (Sigma-Aldrich). HeLa cells were harvested 24–48 h after transfection.
7
Rat Ventral Mesencephalic Cell Culture
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Ventral mesencephalic tissue was dissected from rat embryos of 14 days of gestation (E14). The tissue was incubated in 0.1% trypsin (Sigma), 0.05% DNase (Sigma, St. Louis, MO, USA) and DMEM (Invitrogen Life Technologies, Paisley, Scotland, UK) for 20 min at 37°C, and was then washed in DNase/DMEM and mechanically dissociated. The resulting cell suspension was centrifuged at 50 g for 5min, the supernatant was then removed carefully and the pellet was resuspended in 0.05% DNase/DMEM to the final volume required. The number of viable cells in the suspension was estimated by acridine orange/ethidium bromide staining, and cells were plated onto 35-mm culture dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA) previously coated with poly-L-lysine (100 μg/ml; Sigma) and laminin (4 μg/ml; Sigma). The cells were seeded at a density of 1.5 × 105 cells/cm2 and maintained under control conditions [DMEM/HAMS F12/(1:1) containing 10% fetal bovine serum (FBS; BiochromKG, Berlin, Germany)]. The cell cultures were maintained in a humidified CO2 incubator (5% CO2; 37°C) for 7 days in vitro (DIV); the medium was totally removed on day 2 and replaced with fresh culture medium.
8
Synchronization and Luciferase Assay for Fibroblasts
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For mouse cell experiments, fibroblasts cultured in 35-mm culture dishes (Falcon) were synchronized either with serum shock (50% horse serum for 3 hours) or with temperature entrainment (cycles of 16 hours at 35°C and 8 hours at 37°C for 5 days). During recording, cells were cultured in phenol-free DMEM (Gibco) containing 10% fetal bovine serum (FBS), 1% PSG, and 0.1 mM luciferin, sealed with parafilm to avoid evaporation, in the LumiCycler setup (Actimetrics) at 37°C and 5% CO2. NIH/3t3 murine fibroblasts were cultured under the same conditions as the immortalized fibroblasts but synchronized with 100 nM dexamethasone treatment for 15 min. SMG1 inhibitor (hSMG-1 inhibitor 11e; ProbeChem catalog no. PC-35788) (38 (link)) was used as 10 mM stock (dissolved in dimethyl sulfoxide) and, if not indicated otherwise, used at a concentration of 0.6 μM (NIH/3t3 experiments) to 1 μM (Smg6flox fibroblasts).
9
Generation of CRISPR-Edited Per2 Knockout mESCs
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On day 0, Per2(−/−) ES cells (5 × 105) were plated in 2.0 mL ES medium 2i(+)LIF(+) on 35-mm culture dishes (Falcon) that had been coated with 0.2% gelatine. Five hours after plating, cells were transfected with 1 μg targeting vector, 2 μg pCAGGS-ROSA-TALEN-N153C63-R, and 2 μg pCAGGS-ROSA-TALEN-N153C63-L using Xfect Stem mESC reagent (Clontech) according to the manufacturer’s protocol with the modifications described in the Supplemental Methods . On day 3, cells were harvested and seeded at 1 × 106 on 0.2% gelatine-coated 60-mm dishes. Selection with 1.2 μg/mL puromycin (Sigma) was carried out for 24 h on days 4 and 6. ES clones were picked on or after day 8 as described in the Supplemental Methods . Cells were plated in ES medium 2i(+)LIF(+) on 0.2% gelatine-coated 24-well plates (Techno Plastic Products). Approximately one third of the cells were lysed and used in PCR screening.
10
Mesencephalic Primary Culture Protocol
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Mesencephalic primary cultures were obtained by dissecting ventral mesencephalic tissues from rat embryos of 14 days of gestation (E14). After an incubation of 20 min at 37 °C with 0.1 % of trypsine, 0.05% of DNase (Sigma), and DMEM (Invitrogen), the tissue was washed and mechanically dissociated in DNase/DMEM. The cell suspension was centrifuged at 50×g for 5 min, and the resulting pellet was resuspended in 0.05 % DNase/DMEM. Cells were plated at a density of 1.5 × 105 cells/cm2 onto 35-mm culture dishes (Falcon) previously coated with poly-l -lysine (100 μg/ml; Sigma) and laminin (4 μg/ml; Sigma), and maintained under control conditions (DMEM/HAMS F12/(1:1) containing 10 % fetal bovine serum (FBS) in a humidified CO2 incubator (5 % CO2; 37 °C) for 8 days in vitro (DIV); the entire culture medium was removed on day 2 and replaced with a fresh culture medium.
The dopaminergic cell line N27 (SCC048, Millipore, MA, USA) was cultured in RPMI 1640 medium supplemented with 10 % FBS, 2 mMl -glutamine (Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin. Human embryonic kidney 293 cells, HEK293 (CRL-11268, ATCC), were cultured in DMEM medium supplemented with 10 % FBS, 2 mM l -glutamine (Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin.
The dopaminergic cell line N27 (SCC048, Millipore, MA, USA) was cultured in RPMI 1640 medium supplemented with 10 % FBS, 2 mM
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