Cell proliferation assays

5x10³CFSC in 100 μl 10% FBS were seeded into a 96-well flat-bottom tissue culture plate (Nunc, Roskilde, Denmark). After 24 hours, medium was completely removed, and cells were rinsed with 200 μl phosphate-buffered saline and 100 μl standard medium containing only 0.25% FBS were added. Cells were cultured for additional 24 h prior to treatment. Synchronized microcultures in 96-well plates were treated for 2 hours in reduction medium. When appropriate, the reduction medium was supplemented with 40 mg/l of α1(VI), α2(VI) or α3(VI). Then, 30 mg/l CVI or CI as control were added and cells were incubated for 24 h after which [³H]-thymidine was added for additional 24 h. To assess effects of α3(VI)-derived 30-mer peptides on CVI-induced proliferation of CFSC, the reduction medium was supplemented with a 100-fold molar excess of the respective 30-mer peptides compared to CVI. [³H]-thymidine incorporation was measured as described before [20]. Cells treated with 150 mmol/l HAc or grown in 10% FBS served as negative and positive controls, respectively. During the last 4 hours of treatment, all cultures received 18.5 kBq [³H]-thymidine (Amersham, Freiburg, Germany). Cells were fixed with 10% trichloroacetic acid and lysed by 200 mmol/l NaOH. After neutralization with 800 mmol/l HCl, lysates were collected to a glassfibre mat and the de-novo DNA synthesis was assessed by liquid scintillation (LKB Wallac, Bromma, Sweden) and the radioactive decay was counted (for) a minute (cpm).