Keratinocyte-Trichophyton rubrum Coculture

Keratinocytes were collected, washed, and counted in a hemocytometer. A total of 2.5 × 10⁵ cells/mL were plated on RPMI plus 2% FBS in 250 mL tissue culture flasks and cultured for 24 h at 37°C in 5% CO₂. The T. rubrum solution (1 × 10⁷ conidia/mL) was grown in 5 mL liquid Sabouraud medium for 7 h under gentle shaking. The keratinocyte culture was recovered by centrifugation, washed in saline, and resuspended in RPMI plus 2% FBS. The conidia solution and 0.288 μM trans-chalcone or 4.32 μM α-solanine or 0.0162 μM terbinafine were added to the keratinocyte cultures and incubated for 24 h at 37°C in 5% CO₂. One control and one coculture flask per treatment were reserved for staining with May-Grünwald and Giemsa for verification of infection. Fungi and human cells were recovered by scraping and centrifuged at 1,730 g for 10 min. RNA was prepared directly from the recovered cells as described in the next item.