P2 cerebellar mixed primary cell cultures were grown on poly-L-lysine and laminin-coated (Sigma-Aldrich, MO, USA) coverslips and were grown in 10% foetal bovine serum in IMDM medium (all from Gibco, Invitrogen).
Cells were counted and resuspended in IMDM supplemented with 10 % FBS and 1 % penicillin/streptomycin (full medium) (all from Life Technologies).
Cells were cultured in DMEM medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (complete medium, all from Invitrogen, Carlsbad, CA, USA).
Cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (DMEM complete medium; all from Invitrogen, Gaithersburg, MD, USA) in tissue culture flasks.
Cells were maintained in low-glucose (1 g/L) Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (low endotoxin), 2 mM L-glutamine, and 100 U/ml penicillin-streptomycin (DMEM complete medium, all from Biochrom, Berlin, Germany).
The samples were cultured aerobically at 37 °C for 48 hours on blood plates (Colombia agar with 5% sheep blood), chocolate agar, and CHROMagar��� Candida medium (all from Becton-Dickinson, Franklin Lakes, NJ, USA), as well as anaerobically using Schaedler agar (5% sheep blood, bioMerieux, Marcy l'Etoile, France).
Cells were cultured in RPMI 1640 growth medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% l-glutamine, and 2.5 μg/mL gentamicin (complete medium; all purchased from Invitrogen, CA, USA).
Precipitation was also not evident when proteins were tested against other cell culture media (Roswell Park Memorial Institute 1640 or RPMI-1640, F12 medium, medium 199, Glascow minimum essential medium, and Leibovitz L-15 medium, all obtained from Gibco; data not shown).
ES cells were trypsinized and plated at a density of 30,000 cells cm-2 onto non-coated flasks in neurobasal medium containing 2 mM L-glutamine, B27 supplement and 100 U/ml penicillin-streptomycin (NB27 medium; all reagents from Invitrogen Corp ., plus 1000 U/ml LIF, 10 ng/ml FGF2 and 50 ng/ml EGF (Promega).
Briefly, single cell suspensions were seeded on collagen-coated plates in Dulbecco's MEM/Ham's F-12 (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin (complete culture medium; all reagents from PAA Laboratories, Pasching, Austria) at 37°C in a 5% CO2 humidified atmosphere.
Cells were maintained in knockout serum replacement (KSR) medium (all components were from Invitrogen unless otherwise stated): Dulbecco's modified Eagle's medium/F12 containing 20% knockout serum replacement, fibroblast growth factor 2 (FGF2) (4 ng/ml) (PreproTech), 1 mM L-glutamine, 1 mM non-essential amino acids, 1 mM 2-mercaptoethanol, penicillin (50 U/ml), and streptomycin (50 µg/ml).
