Wound healing

Rinse flasks, trypsinize cells to detach them, inactivate trypsin with complete media, count cells using trypan blue exclusion, and plate cells in 24-well plates. Incubate the plate to allow cells to attach and form a confluent monolayer.

Disinfect all materials with 70% ethanol. Pre-made capsaicin serial dilutions in 15 ml conical tubes are ready for use along with control treatments (RPMI with reduced serum, ceramide, and DMSO vehicle control).

Remove culture media from wells and replace with PBS to prevent cell death during preparation. Draw black marker lines across all wells perpendicular to the wells to serve as a consistent reference point for imaging the wounds at each time point.

Add PBS to rehydrate wells, then use 200 microliter micropipette tips to scratch perpendicular to the black marker lines, creating consistent wound gaps in the cell monolayer. Standardize pressure and angle to maintain consistent wound widths across all wells.

Aspirate PBS to remove dead cells and debris from scratched wells. Add regular RPMI culture media with reduced 5% FBS (instead of 10%) to control wells, ceramide as a positive control to inhibit migration, and DMSO vehicle control to another set of wells.

Remove PBS from individual treatment groups and replace with five different concentrations of capsaicin ranging from 2 micromolar to 33 micromolar in low-serum RPMI media. Proceed through each concentration group individually to prevent cell drying.

Take photographs of all wound sites at time point zero using the black marker lines as reference to ensure consistent imaging position. These baseline images will be used to compare wound closure across all treatment conditions.