Keratinocyte Dissociation Assay for Pemphigus Vulgaris

A keratinocyte dissociation assay is currently the main tool for the analysis of antibody-induced acantholysis in PV in vitro [26 (link)], and it was performed as previously described in [24 (link)]. In brief, HaCaT cells were cultivated in 24-well plates with DMEM containing GlutaMAX (Gibco, Grand Island, NY, USA) and CaCl₂ (1 mM) in a controlled atmosphere (CO₂ 5%) at 37 °C. Twenty-four hours after reaching the confluence, positive control (AK23: 10 µg/mL), purified IgG from HD (62.5 µg/mL) or from PV patients (62.5 µg/mL) were added and incubated for 24 h. The HaCaT cells were then treated with a dispase solution (2.4 U/mL; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C until the monolayers were separated from the plate. The monolayers were stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) and mechanical stressed by pipetting 7 times with a 1 mL pipette. After the fixation of cell fragments, pictures were taken, and the number of fragments were counted manually. Each experiment was performed in triplicate.