- Thaw mast cells following standard procedure and wash once.
- Adjust cell concentration to ≥200,000 cells per mL.
- Add IL-3 to the medium at 100 U/mL and SCF at 50 ng/mL.
- Culture cells in a standard T75 flask at 37°C, 5% CO2.
- Cells grow in suspension and may form cell clusters.
- Split cells when the culture reaches a density of 3-5 x 106 cells per mL. The medium may appear orange-yellow.
- Pipet the culture medium up and down vigorously to break up the cell clumps. Adjust cell concentration by adding fresh medium containing cytokines.