After 24 hours, the medium was discharged and the formation of formazan was measured by adding MTT (5 mg/ml in PBS, 50 µg/well) and incubating the wells for 3 hours in the dark at 37°C.
After centrifugation, the medium was discharged and the intact cells re-suspended partly in a buffer containing 0.75% (v/v) glycine and 0.25% (v/v) bovine serum albumin adjusted to pH 8.8 for insulin determination after sonication and centrifugation at 30,000×g for 30 minutes at 4°C, and partly in 0.1 mol/l NaOH for protein determination.
After these incubation times, the medium was discharged, the cells were washed twice with PBS and 120 μl PBS was added to cells.
After incubation for 20 h the medium was discharged, the cells were washed twice with PBS and new medium containing 10 μM DCF or, as control, medium without DCF was added to cells and incubation was continued for 1 h.
Culture medium was discharged and differentiation medium containing DMEM/Ham's F12, 2% donor horse serum (Biochrom AG, Berlin, Germany), 1% L-glutamine, 1 ng/mL bFGF (Sigma Aldrich, St . Louis USA), and 0,4 μg/mL dexamethasone (Sigma Aldrich, St . Louis USA) was added to cell culture dishes as described previously [ 4].
Thereafter, the medium was discharged again, the cells were washed twice with PBS and new medium containing 30 μM FeCl3, 30 μM FeCl3/30 μM InsP6 or 30 μM FeCl3/100 μM InsP6 was added and the cells were further incubated for 2 or 20 h, respectively.
HUVECs were harvested at the designated time points, and RNA was extracted and quantified for Microarray analysis (Affymetrix measurement) The medium with insulin was collected and stored at −20°C for the insulin detection (Insulin Myria, Technogenetics), while the medium without insulin was discharged.
He was discharged in January 2013.
He was discharged on Tuesday.
"She was discharged last night".
