Isolation of dermal mast cell (DMC)

1. Human DMC were isolated from skin by enzymatic digestion.

2. Dermal tissue was obtained from patients with skin after informed consent was given.

3. The skins were resected and placed into Ca/Mg-free primary cell buffer.

4. Tissue was chopped into small pieces, and the fragments were washed extensively in primary cell buffer.

5. Tissue was incubated with primary cell isolation kit for 2 hours at 37°C and the dispersed cells were recovered by filtration through Nytex cloth.

6. After washing twice in 0.9%NaCI, the cell suspension was incubated with primary cell isolation kit for 15 minutes at 37°C to remove (skin) cell ghosts.

7. Cells were washed three times in NaCl at room temperature.

8. The MCs was determined by primary cell identification kit.

9. Isolated MC were cultured in primary cell culture system supplemented with 10% fetal calf serum (FCS), glutamine, and antibiotics at 37°C for at least 24 hours before being analyzed.

10. In initial experiments, MCs were isolated from various sites of the skin.