Hi! I am trying to setup a basophil activation test in allergic patients for a study. As I try it for the first time, I have a few questions:

1)As we will have several patients a day (and various concentrations of allergens), I plan to do it in 96 well plates and read it also in the plates in a Beckmann Coulter flow cytometer. Does any of you have experience with that?

2) Could anyone provide me with a detailed protocol how long to incubate the cells with the allergen, in which buffer, if you add some solution to stop or which fixing solution you use, etc.? I tried to find something in the web, but its not so easy...is it feasible to do it in 96 well plates?

3) What are your thoughts on which antibodies to use for identification of basophils (CD123+HLADR or CCR3?) or for activation (CD203c or CD63 or both?)?

Any help would be very much appreciated!

Thanks! Julia