Dispase-based dissociation assay

NHEKs were grown to confluence in triplicates on 6-well plates and exposed to PV or normal serum in a 1:10 dilution or to PV or control IgG antibodies in a final concentration of 65 μg/ml. After 24h the cells were washed twice with PBS, incubated in 2 ml of dispase II (2.4 units/ml, Roche Diagnostics, Basel, Switzerland) at 37°C for 40 minutes and detached from the plate as monolayers. Cell sheets were carefully transferred to 15 ml tube containing 5 ml PBS and subjected to mechanical stress using 5 inversions. The number of fragments was counted by two independent evaluators.