Shreffler: Buhlmann PNOIT

Overview

Basophil activation test done using assay kit provided by Buhlmann/Alpco immunoassays. Assay calls for use of peripheral blood, and is quick and dirty compared to other basophil stimulation protocols (completely fine due to qualitative rather than quantitative nature of BAT).

This protocol is modified to use serum-removed cell samples.

Materials

• Flow2 CAST kit provided by Buhlmann/Alpco (FK-CCR, Buhlmann)
• Allergens of interest, provided by Buhlmann/Alpco
• Lavender top k-EDTA tubes (2) from clinic
• Gold top clot activator tube (1) from clinic
• 5 mL polypropylene tubes (Falcon, 352002)
• 5 mL polystyrene tubes for flow cytometer acquisition (Falcon, 253008)
• Ca/Mg-free PBS
• DI water

Preparation of supplied reagents

• Stimulation Buffer (B-CCR-STB) - reconstitute w/ 50 mL DI water
• Stimulation Control (anti-FceRI mAb) (B-CCR-STCON) - reconstitute w/ 1.5 mL stim. buffer
• Stimulation Control (fMLP) (B-CCR-FMLP) - reconstitute w/ 1.5 mL stim. buffer
• Lysing Reagent (B-CCR-LYR) - dilute w/ 225 mL DI water

Procedure

• For more detailed information, refer to Flow2 CAST guidebook included in Buhlmann kit

1. Spin gold-top tube at 500 g for 10 min. Collect serum in sterile 5 ml polystyrene tube w/ cap and parafilm after closing. Store at -20°C.
2. Tube 1 of the lavender-top tubes will be used as whole blood and tube 2 will be used as'washed' blood cells. Spin both tubes at 500 g for 7 min. Aspirate and discard plasma layer from tube 2, and restore to original volume w/ calcium free PBS. Gently resuspend cells in both tubes by inverting.
3. Label PP tubes as follows (make two sets of tubes; one prime ['] set for plasma-removed samples):
   • PB = patient background
   • PC1 = stimulation control with anti-FCeRI ab
   • PC2 = stimulation control with fMLP
   • desired allergen panel/dilutions (for PNOIT, please prepare the following):
     • Buhlmann allergens at five 5-fold dilutions each (see piptting scheme here:Media:Image-110217 Peanut F2C pipetting scheme.xls - resuspend lyophilized allergen in 250 µl of stim buffer; use this as top conc. and make 1:5 dilutions starting here)>
       • Arah1
       • Arah2
       • Arah6
       • F13 Peanut Lot 0503
     • Two special Arah-free peanut extracts at five 5-fold dilutions each.
     • CDE at four 10-fold dilutions starting at top concentration of 3 BAU (10% of 30 BAU stock).
4. Add 100 µL stimulation buffer to each tube.
5. Add 50 µL of each stimulant to appropriate tube
   • PB tube: stimulation buffer (background)
   • PC1 tube: stimulation control (anti-FCeRI ab)
   • PC2 tube: stimulation control (fMLP)
   • Allergens: refer to following pipetting schemes:

1. Add 50 µL of patient's whole blood to each tube
2. Add 20 µL staining reagent to each tube (cocktail of CCR3/CD123-PE and CD63-FITC, included in kit)
3. Mix gently by tapping each tube with finger
4. Incubate at 37ºC for 15 minutes if in water bath, 25 minutes if using incubator
5. Add 2 mL pre-warmed (18-28ºC - room temperature) lysing reagent to each tube, mixing gently on vortex while adding lysing reagent
6. Incubate for 10 minutes at RT
7. Centrifuge tubes for 5 minutes at 500 x g
8. Decant supernatant by pouring into sink with one swift motion, making sure not to disturb/resuspend pellet
9. Transfer resuspended samples to labeled PS tubes for data acquisition on flow cytometer. Samples can be stored for a few hours protected from light and at 2-8ºC, but acquisition should be done within the same day