When switching to a new batch of any medium component (including serum), even though this has been batch-tested, retain a reasonable amount of the old batch for some period of time.
Culture medium and supplements including serum were from BioWhittaker (Walkersville, MA).
To complete differentiation the cells were then cultured for further 12 days with adipocyte nutrition medium (including fetal calf serum 3%, d-Biotin 8 μg/mL, Insulin 0.5 μg/mL, Dexamethasone 400 pg/ml).
Caco-2, HT-29, and SW480 cells were cultured in Roswell Park Memorial Institute-1640 Institute-1640 RPMI-1640 RPMI-1640l bovine serumedium) and 1% (v/v) penincluding10,000 U/ml)/streptomycin (10,000 U/ml) (P/S).
After 7 days in cell culture medium including fetal calf serum (FCS), 50 μg/mL endothelial cell growth supplement, 30 U/mL heparin, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM alanine-glutamine HGMEC constituted approx. 10% of glomerular outgrowths.
The tissues were minced and dissociated and then grown in cultures with Neurobasal medium including 12% horse serum, 0.6% D-glucose and 2 mM L-glutamine on 8-well chamber slides (Falcon, Franklin Lakes, NJ, USA) coated with 5 μg/ml poly-D-lysine at 37°C in a humidified incubator with 10% CO2.
The reaming debris was cultured in Petri dishes with F12 K medium including 20 % foetal calf serum (FCS), 100 U/ml penicillin and 100 μg/g streptomycin.
In brief, the culture medium containing the test substances was replaced with fresh serum-containing medium including 60 μg/ml of Neutral Red (Carl Roth, Karlsruhe, Germany).
To assess the effects of SPARC on FGF-induced proliferation, cells (10 000 per well) were propagated for 48 h in serum-free medium including the indicated factors.
