Denaturation Study

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)

 Protocol

  1. Collect fresh blood from a Der p1 sensitive individual in heparinized tubes
  2. Mix by inversion
  3. Prepare dilution series for extracted allergen in stimulation buffer – supplied (1:10. 1:100, 1:1000)
  4. Add 100ml of allergen dilution to 1.5ml tube followed by 100ml of heparinized whole blood.
  5. 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.
  6. Incubate for 20 minutes at 37°C
  7. Add 20ml of staining reagent and incubate for 20 minutes on ice.
  8. 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)
  9. Remove supernatant and add resuspend cells in 2ml of PBS – o.5% BSA
  10. Wash by centrifugation at 540 x g for 5 minutes
  11. Remove supernatant and resuspend in 300ml of PBS buffer.

Analysis

Positive basophils gated on   

  • CD123 PE-A positive
  • CD63 FITC-A positive

 

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