Mast cell activation and generation of supernatant samples
After 8 weeks of culture, IgE sensitized mast cells were activated by
challenge with recombinant Der p 2 allergen in log10dilutions for 30 minutes in a water bath at 37° C. Activation
was assessed by flow cytometry as surface expression of CD63.
Supernatants were generated by activating MCs for 3, 6 or 24 hours at
37° C with an optimal concentration of Der p 2 allergen
determined above. After centrifugation for 10 minutes, 4° C,
2000G, the supernatant and pellet were frozen separately. A supernatant
sample not activated with allergen (time 0 hours) was saved as baseline
control.