Primary myoblasts were isolated from hindlimb muscles of C57BL/6 mice. Muscle tissue was minced and digested with 0.1 mg/ml collagenase II (Sigma Aldrich Co, St. Louis, MO) for 20 minutes and 1 mg/ml collagenase/dispase (Roche Life Science, Indianapolis, IN) for 30 minutes at 37°C. The homogenate was filtered sequentially in 100 μm, 70 μm and 40 μm nylon mesh cell strainers (Fisher Scientific, Waltham, MA), and centrifuged at 1,200 rpm for 10 minutes. The cell pellet was suspended in 10 ml of plating media (DMEM supplemented with 10% horse serum (Sigma, St. Louis, MO), 0.5% chicken embryo extract (CEE) (MP Biomedicals, Santa Ana, CA), 4 mM L-glutamine, and 1% penicillin/streptomycin solution. Cells were pre-plated in the incubator (5% CO2, 37°C) for 1 hour to remove fibroblasts. The enriched myoblast solution was transferred in a 100 mm tissue culture dish coated with collagen (BD Bioscience, San Jose, CA). After 48 hours, the medium was changed to fresh growth medium: DMEM supplemented with 20% fetal bovine serum, penicillin/streptomycin (50 U/ml/50 mg/ml), L-glutamine (4 mM), hepes (10 mM), and CEE (3%).
Tissue culture dish
Manufactured by BD
Sourced in United States
The tissue culture dish is a type of laboratory equipment used for the in vitro cultivation of cells, tissues, or organisms. It provides a controlled environment for the growth and maintenance of cell cultures.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
1 n nitric acid solution, by Fujifilm (2 mentions)
Nylon mesh cell strainer, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Merck Group (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
Collagenase dispase, by Roche (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem low glucose, by Merck Group (1 mentions)
L ascorbic acid, by Fujifilm (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Fetal bovine serum fbs, by Nichirei Biosciences (1 mentions)
Poly hema, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Cyquant ldh cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Clariostar, by BMG Labtech (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
10 protocols using tissue culture dish
1
Isolation of Primary Mouse Myoblasts
2
Isolation and Culture of UC-MSCs
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Human umbilical cords (n=3) were purchased from StemExpress (Placerville, CA, USA). We isolated and purified UC-MSCs according to the protocol previously published by our group [22 (link)]. In brief, the vessels of UC were removed to retain Wharton's jelly. Wharton's jelly tissue was then cut into 1 mm3 pieces, added to the bottom of the tissue culture dish (BD, Franklin Lakes, New Jersey), and incubated at 37°C in a 5% carbon dioxide incubator without culture medium for two hours to allow tissue attachment. 15ml complete culture medium containing DMEM (low glucose) supplemented with 10% FBS (fetal bovine serum), 1% L-glutamine, and 1% penicillin-streptomycin (all from Invitrogen, Grand Island, NY) was added to the tissue culture dishes and incubated for an additional 7 days at 37°C and 5% carbon dioxide incubator. The purified Wharton's jelly pieces were removed and the primary cells that had migrated out of the Wharton's jelly tissues and attached to tissue culture dishes were passaged by a 1-min treatment with 0.25% trypsin and 0.02% EDTA at 37°C. The medium was changed every 3 days. UC-MSCs were further passaged when they reached 90 confluency. All UC-MSCs used in experiments were passages 3-5.
3
Osteogenic Differentiation of Murine BMSCs
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After euthanasia, femurs and tibiae of 6–9 week-old wild-type and Runx2+/− mice were carefully cleaned from adherent soft tissue and bone marrow cells were harvested. Collected cells were seeded at a density of 4 × 107 cells per 3.5 cm tissue culture dish (BD Falcon) and cultured in growth medium: Dulbecco's modified Eagle's medium-low glucose (DMEM; Sigma) containing 10% heat-inactivated fetal bovine serum (FBS; Nichirei) and penicillin/streptomycin (100 IU/ml and 100 μg/ml; Sigma), at 37 °C in 5% CO2 atmosphere. After 4 days of culture, nonadherent cells were removed and adherent cells were cultured 3 more days until 90% confluence to use as BMSCs in this study. For promoting osteogenesis, BMSCs were cultured in osteogenic medium: growth medium supplemented with 10 nM dexamethasone (Sigma), 82 μg/ml l -ascorbic acid (Wako) and 10 mM β-glycerophosphate (Sigma), at 37 °C in 5% CO2 atmosphere. Osteogenic medium was changed every three days.
4
Culturing Adherent and Suspension Cancer Cells
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MCF-7 and MDA-MB-231 human breast tumor cells and A549 human lung tumor cells were cultured in Dulbecco's modified Eagle's medium (GibcoBRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GibcoBRL). Adherent cells were cultured in a tissue culture dish (Falcon, San Jose, CA, USA), while suspension cultures were grown in dishes coated with 10 mg/mL poly(2-hydroxyethyl methacrylate) (poly-HEMA, Sigma-Aldrich). To select cells that could survive in suspension culture, 1×106 cells were seeded onto poly-HEMA-coated dishes and incubated for more than a week for use in further experiments. Fresh medium was added every three days. After seven days in culture, cells were harvested and treated with diluted trypsin-EDTA to obtain a single-cell suspension for re-plating or Trypan blue dye exclusion assays.
5
Cell Viability Assay for NMP Inhibitor
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To verify that the NMP inhibitor does not affect cell viability, cytotoxicity was measured by using CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen). THP-1-derived macrophages were grown in RPMI with or without NMP (100 µg/ml) in tissue culture dish (35 X 10 mm, Falcon) at 1.0 × 106 cells/well in growth medium and LDH activity in the medium was determined after 2- or 4-h treatment following manufacturer instructions. Samples containing maximum LDH activity and spontaneous LDH activity were also prepared as well as the LDH positive control. The absorbance has been measured at 490 nm and 680 nm with a CLARIOstar plate reader (BMG LABTECH, Offenburg, Germany) and the percentage of cytotoxicity was calculated according to the manufacturer’s instructions.
6
Murine L929 Cells Maintenance Protocol
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Murine L929 cells (ATCC, CCL-1, Manassas, VA) were maintained in Dulbecco’s Modified Eagle Medium High Glucose Plus (DMEM, LGC Biotecnologia, BR-30356-05, Cotia, Brazil) containing 1 mM sodium pyruvate and 25 mM glucose and supplemented with 10% (v/v) heat-inactivated Horse Serum, New Zealand Origin (Gibco, 16050122, Waltham, MA) at 37°C and 5% (v/v) CO2 according to the manufacturer’s instructions. L929 cells were seeded in a 150 mm diameter Tissue Culture Dish (Falcon, 353025, Corning, NY) and allowed to grow until 80% area confluence. Then, the medium was completely replaced and the cells were incubated with 50 ml of DMEM (Vitrocell, D0069, Campinas, Brazil) containing 25 mM glucose, 1 mM sodium pyruvate, 4 mM L-glutamine, and 2% (v/v) heat-inactivated fetal bovine serum (Vitrocell, 11) for 3 days at 37°C and 5% (v/v) CO2. This L929 conditioned medium was collected, sterilized using a 0.22 µm filter, and frozen at −20°C before use.
7
Quantifying NHEJ Repair and Translocation
8
In Vitro 10B Uptake Study
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For the in vitro 10B uptake study, B16 mouse melanoma cells, C6 rat glioma cells, and F98 rat glioma cells were used. B16 and C6 cells were used for in vitro testing at Osaka Prefecture University. Based on results from initial in vitro screening, the in vitro 10B uptake study using F98 glioma cells were performed at Osaka Medical College. Four hundred thousand cells were seeded in a tissue culture dish (100 × 20 mm; Becton Dickinson, Franklin Lakes, NJ, USA) with DMEM supplemented with 10% FBS and 10% penicillin and streptomycin at 37 °C in a 5% CO2 atmosphere. After incubation for 4 days at 37 °C, the medium was removed, and DMEM with 10% FBS containing 1 mM KA-BSH, BSH, or BPA was added to each dish. The cells were incubated for an additional 24 h at 37 °C. The medium was then removed, and the cells were washed twice with 4 °C phosphate-buffered saline (PBS) and detached with trypsin–ethylenediamine tetraacetic acid solution. PBS was then added, and cells were digested overnight with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan), and 10B uptake was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) using an iCAP6000 emission spectrometer (Hitachi High-Technologies, Tokyo, Japan).
9
Boron Uptake in Rat Glioma Cells
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The intracellular 10B concentrations were measured by means of inductively coupled plasma atomic emission spectroscopy (ICP-AES). For the in vitro boron uptake studies, F98 rat glioma cells were used. First, 105 F98 glioma cells were seeded into a tissue culture dish (100 × 20 mm; Becton Dickinson, Franklin Lakes, NJ) with culture medium (described above) at 37 °C in a 5% CO2 atm. After incubation for 5 days at 37 °C, the medium was replaced with culture medium (described above) containing 1 mM of ACBC-BSH, BSH, or BPA, and the cells were incubated for an additional 24 h at 37 °C. The medium then was removed, and the cells were washed twice with phosphate-buffered saline (PBS) and detached with trypsin-ethylenediamine tetraacetic acid solution. PBS was then added, and the cells were centrifuged twice and counted and sedimented.
The cells were then digested for 1 week with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan), and the boron uptake was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES: Hitachi, Tokyo). Pairwise comparisons were conducted using Student’s t-test. Group differences resulting in p-values < 0.05 were considered significant.
The cells were then digested for 1 week with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan), and the boron uptake was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES: Hitachi, Tokyo). Pairwise comparisons were conducted using Student’s t-test. Group differences resulting in p-values < 0.05 were considered significant.
10
Compartmented Culture for Axon Guidance Studies
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