PriCells: Primary cell attachment assays

1. 96-wells tissue culture plates were coated for 1 hour at RT with type I collagen, washed several time and blocked with PriCells culture solution and 1% BSA for an additional 30 minutes.

2. Primary cell were trypsinized off flasks, centrifuged and resuspended in PriCells culture system with 1% BSA.

3. The block was washed with PriCells culture solution and wells filled with 5×104 cells/well in PriCells culture solution and 1% BSA.

4. Cells were allowed to attach for 1 hour at 37°C, as determined by preliminary time course experiments.

5. Unattached cells were discarded and washed three times with PriCells culture solution and 1% BSA.

6. Attached cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 1% Methylene Blue in 0.01 M borate for 30 minutes.

7. Excess dye was washed off with water and bound cells were lysed with EtOH/0.1 M HCl (1:1).

8. The absorbance of released dye was read at 650 nm on a multiwell plate reader.