Culture flask were coated with DMEM (glucose 4.5 g/L; Gibco, USA) containing PS (1%; Gibco, USA), fibronectin (2 μg/mL; Gibco, USA), and extracellular matrix (1% vol/vol; Sigma, USA) and incubated for at least 1h in 5% CO2 at 37°C before the cells were seeded. EndoC-βH1 cells were grown on Matrigel/fibronectin-coated culture flasks containing DMEM (glucose 1 g/L), BSA fraction V (2% wt/vol) (Roche, Germany), 2-mercaptoethanol (50 μM; Sigma, USA), nicotinamide (10 mM; Sigma, USA), transferrin (5.5 μg/mL; Sigma, USA), sodium selenite (6.7 ng/mL; Sigma, USA), PS (1%)16 (link).
Extracellular matrix
Manufactured by Merck Group
Sourced in United States, Germany
Extracellular matrix is a laboratory equipment used for the study and manipulation of the extracellular environment. It provides a three-dimensional scaffold that supports and regulates cellular behavior, including adhesion, migration, proliferation, and differentiation. The core function of the extracellular matrix is to mimic the natural physiological environment, enabling researchers to investigate cellular processes and develop novel therapeutic approaches.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Nicotinamide, by Merck Group (3 mentions)
Transferrin, by Merck Group (3 mentions)
Sodium selenite, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fibronectin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Roche (2 mentions)
Bsa fraction 5, by Roche (2 mentions)
Fibronectin, by Roche (2 mentions)
L glutamine, by Merck Group (2 mentions)
Giemsa solution, by Solarbio (2 mentions)
24 well transwell chamber, by Corning (2 mentions)
24 well plates chamber, by Corning (1 mentions)
Upright light microscope, by Olympus (1 mentions)
Endothelial cell medium (ecm), by Merck Group (1 mentions)
Transwell chamber, by Corning (1 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
21 protocols using extracellular matrix
1
Culturing Insulin-Producing EndoC-βH1 Cells
2
Trans-well migration assay for Caki-1 cells
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Caki-1 cells, which were infected with control or miR-30c, were analyzed by trans-well assays performed in 24-well plates chamber (Corning Incorporated) fitted with a polyethylene terephthalate filter membrane with 8-µm pores. Next, the membrane’s undersurface was preincubated by 30 µL ECM (Extracellular matrix) from Sigma-Aldrich Co., mixed with serum-free RPMI 1640 in 1:5 dilution for 4 h at 37°C. The top chambers of the trans-wells were filled with 0.1 mL of cells (5×105 cells per mL) in serum-free medium, and the bottom chambers were filled with 0.3 mL of RPMI 1640 containing 10% FBS. The cells were incubated in the trans-wells at 37°C in 5% CO2 for 12 h. For migration assay, the cells were incubated in chambers without an ECM coating.
3
Transwell Migration and Invasion Assay
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Transwell chambers (Corning, USA), with 8 μm of pore size, were employed to assess migratory and invasive capabilities of hepatocarcinoma cells. Additionally, ECM (extracellular matrix, Sigma, USA) with dilution 1:50 in serum-free medium was pre-coated in chambers for invasion experiment. After the transfections of si-AFAP1-AS1 and si-NC, 10,000 cells from each group were suspended in 1 ml media with FBS, DMEM for HCCLM3 and RPMI-1640 for HepG2. Then, 200 μl of both calculated cells were charged into upper chamber of the unit and mounted on top of each well of a 24-well plate. Subsequently, 600 μl medium with 20% FBS, acting as cell’s chemo attractants, was added into each of the respective bottom chamber, then incubated for 48 h at 37 °C with 5% CO2. The migrated or invaded cells on lower surface of filter were subjected for 0.5% crystal violet staining after fixing in absolute methanol. Then five fields of lower sides of chambers were captured using upright light microscope (Olympus, Japan), with 20 × of magnification.
4
Human Glioma Cell Culture Protocols
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Cells were mycoplasma tested and authenticated. Human glioma cell lines, U87 (RRID:CVCL_0022; https://scicrunch.org/resolver/CVCL_0022 and https://web.expasy.org/cellosaurus/CVCL_0022 ) and U-251MG (RRID:CVCL_2219 https://scicrunch.org/resolver/RRID:CVCL_2219 and https://web.expasy.org/cellosaurus/CVCL_0021 ) (ATCC), were grown in DMEM (Cat# 21969035, Gibco,) supplemented with 10% FBS. Patient-derived glioblastoma cell lines previously isolated from patient samples, as described in,16 (link) were cultured in serum-free Neurobasal Medium (Cat# 12349015, Gibco) supplemented with B27 (Cat#17504044, Gibco), N2 (Cat# A1370701, ThermoFisher Scientific), 20 ng/mL EGF (Cat# E9644, Sigma), 20 ng/mL FGF (Cat # PHG0261, Gibco), 2 mM glutamine and Penicillin-Streptomycin (100 U/ml) (Cat# 15070063, Gibco) in flasks precoated with Extracellular Matrix (Cat# E1270, Sigma). For hypoxia experiments, cells were incubated in a Tri-Gas incubator (1% O2, 5% CO2) or in a hypoxia chamber (0.1% or 1% O2, 5% CO2).
5
Single Fiber Analysis of Dystrophic Muscle
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Injury to whole TA/EDL muscle, single fiber isolation and analysis, muscle histology and immunofluorescence were performed as described previously (Shea et al., 2010 (link)). A longer incubation time (90 min) was required to isolate single fibers from mdx mice. Split fibers that occur as a consequence of disease progression were excluded from the analysis. SCs were fixed immediately or plated in plating medium (DMEM with 10% horse serum) on extracellular matrix (Sigma) for 3-4 days and stained for Pax7, Ki67, MyoD, MyoG, Myf5 and p27kip1 (described in full in supplementary material Table S1).
6
Characterization of EndoC-βH1 Cells
7
Isolation and Culture of Human Muscle Stem Cells
8
Characterization of EndoC-βH1 Cells
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EndoC-βH1 cells provided by EndoCells/INSERM were cultured and passaged as previously described (Ravassard et al., 2011 (link)). Briefly, cells were seeded at a density of approximately 600,000 cells/cm2 on tissue culture-treated plates pre-coated overnight with extracellular matrix (Sigma) and fibronectin (Sigma) in EndoC-βH1 complete medium. Cells were passaged approximately every 7 days. Cells were harvested at various passages and distinct sites (e.g., NHGRI, JAX-GM) for karyotyping, genotyping, ATAC-seq, ChIP-seq, RNA-seq, Hi-C, and Pol2 ChIA-PET analyses.
9
Optimizing FDB Fiber Adherence for Seahorse XF24
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To facilitate fiber adherence to Seahorse XF24 cell culture V7 microplates (Seahorse Bioscience, Mulgrave., Australia), wells were coated with 5 µL of extracellular matrix (Sigma Aldrich, Sydney, AustraliaE1270) diluted in DMEM (1:1). Then, 75 µL of isolated FDB fibers was aliquoted into coated wells and confluency was determined using light microscopy. A minimum of ~60% confluency, i.e., the estimated percentage of the well bottom covered by isolated FDB fibers, was deemed optimal. If a well did not meet this requirement, an additional 50 µL aliquot of fibers was dispensed into wells.
10
Cell Migration and Invasion Assay
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Transfected cells were cultured in the serum‐free DMEM medium on the upper chamber of transwell filter chamber filters that was precoated with Matrigel (Sigma‐Aldrich). DMEM medium containing 10% FBS was put in the lower chamber. Cells were grown in the incubator for 24 h. Afterward, the upper chamber was discarded while cells that migrated to the lower chamber were fixed in 4% paraformaldehyde for 10–15 min at room temperature. 0.2% crystal violet was added to incubate with the cells and then the stained cells were analyzed. To analyze the invasion ability of cancer cells, the upper chamber was precoated with extracellular matrix (Sigma‐Aldrich). Twenty‐four hours later, cells growing in the lower chamber were analyzed with same procedures.
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