Primary astrocyte isolation was performed as described previously [8 (link)]. Briefly, the cortices of newborn Sprague–Dawley rats 1–3 days old were dissociated in DMEM/F12 (1:1) medium (Hyclone Laboratories, Inc., Logan, UT, USA; SH30023.01) and digested in 0.05% trypsin/EDTA (ethylenediaminetetraacetic acid; Gibco, Waltham, MA, USA; 25200056) with 1% DNAse (Worthington Industries Medical Center, Inc., Columbus, OH, USA; IDLS006331) at 37 °C for 25 min to yield a single-cell suspension. Cells were collected and resuspended in DMEM/F12 (Dulbecco’s modified Eagle’s medium/nutrient mixture F-12; 1:1) medium containing 10% fetal bovine serum (FBS; Sigma, Australia, F8318) and plated in culture flasks. After 5 days, the other cell types (oligodendrocytes and microglia) growing on top of the astrocytes were removed by shaking the flasks in an orbital shaker for 16 h. Cells were passaged once they reached over 90% confluence using 0.25% trypsin/EDTA. Cells at passage 1 were used for the experiments.
Dmem f12 1 1 medium
Manufactured by Cytiva
Sourced in United States
DMEM/F12 (1:1) medium is a cell culture medium formulation designed for the in vitro cultivation of a variety of mammalian cell types. It is a balanced salt solution that provides essential nutrients and growth factors required for cell proliferation and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (1 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
Alpha thioglycerol, by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Merck Group (1 mentions)
Penicillin, by Cytiva (1 mentions)
Streptomycin, by Cytiva (1 mentions)
L glutamine, by Cytiva (1 mentions)
2 protocols using dmem f12 1 1 medium
1
Isolation of Primary Astrocytes from Rats
2
Culturing Lung, Mast, and Fibroblast Cells
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The human lung epithelial cell line, A549 (ATCC), was cultured in DMEM/F12 (1:1) medium (HyClone laboratories, Inc., Logan, UT), the human mast cell, HMC-1 (Dr. Joseph Butterfield, Mayo Clinic, Rochester, MN, and a kind gift from professor Gunnar Nilsson at Karolinska Institute, Stockholm, Sweden), was cultured in IMDM (Sigma Aldrich, St. Louis, MO) and the mouse fibroblast cell line, NIH3T3 (ATCC), was cultured in DMEM (with Glutamine; HyClone laboratories, Inc.). All media was supplemented with 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin (HyClone laboratories, Inc.). Additional, 2 mM l-glutamine (HyClone laboratories, Inc.) and 1.2 mM alpha-thioglycerol (Sigma Aldrich) were added to the HMC-1 cell media. All supplement FBS was depleted of EVs by ultracentrifugation for 18 hours at 120,000×g (Type 45 Ti rotor, 38, 800 rpm, k-factor 178.6, Beckman coulter, Brea, CA) unless it is specifically mentioned as non-depleted. All cells were cultured at 37°C and 5% CO2.
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