Staining Reagent Mix
- CD63 FITC (Specific expression in activated Basophils)
- CD203c PE (Basophil and mast cell expression)
- HLA-DR PerCP (Monocytic lineage – not present on Basophils)
- CD123 APC (Expressed on White blood cells – myeloid precursors)
- Collect fresh blood from a Der p1 sensitive individual in heparinized tubes
- Mix by inversion
- Prepare dilution series for extracted allergen in stimulation buffer – supplied (1:10. 1:100, 1:1000)
- Add 100ml of allergen dilution to 1.5ml tube followed by 100ml of heparinized whole blood.
- Use 90ml stimulation buffer with 10ml of fMLP/IgE mix (supplied) and 100ml of stimulation buffer to the negative control follow by 100ml of heparinised blood.
- Incubate for 20 minutes at 37°C
- Add 20ml of staining reagent and incubate for 20 minutes on ice.
- Lysis RBC as per the ammonium chloride lysis method (10 minute incubation at RT with lysis buffer followed by centrifugation at 250 x g for 10 minutes)
- Remove supernatant and add resuspend cells in 2ml of PBS – o.5% BSA
- Wash by centrifugation at 540 x g for 5 minutes
- Remove supernatant and resuspend in 300ml of PBS buffer.
Positive basophils gated on
- CD123 PE-A positive
- CD63 FITC-A positive