DURAClone IF Basophil Activation Assay Protocol

This protocol is for reference purposes only.  It may be necessary to adapt the method for specific research needs.

Materials

KIT BOX CONTENTS

25 tests of the DURAClone IF Basophil Activation Antibody Panel

NOTE: Do not store the reagent tubes in the refrigerator; do not freeze/thaw the tubes. Minimize the exposure of the tubes to light, especially during processing and incubation of sample(s) prior to acquisition.

MATERIAL REQUIRED BUT NOT SUPPLIED

Blood collection tube containing anticoagulant, EDTA

Calibrated pipettes

Vortex mixer

Antigens

Phosphate Buffered Saline, PBS. Prepared according to the IFU prior to use. (Part Number 6603369)

Calcium2+ Activation Solution (Part Number C23407

OptiLyse C Lysing Solution (Part Number A11894)

Flow cytometer equipped with the following lasers and detectors:

  • FSC/SSC
  • 405 nm: 430 – 470 nm, 530 – 570 nm
  • 488 nm: 560 – 600 nm
  • 633 nm: 715 – 735 nm

Sheath fluid

Flow cytometer calibration beads

Staining Procedure 

NO WASH PROCEDURE

  1. Prepare allergen dilutions in activation solution.

  2. Add 50 μL of Activation Solution (with or without allergen) into each tube.

  3. Vortex at high speed for 6 to 8 seconds.

  4. Add 50 μl of blood.

  5. Gently vortex each tube for 1 to 2 seconds and incubate for 15 minutes in a 37 °C water bath. Protect from light.

  6. Remove tubes from the water bath and add 250 μL of OptiLyse C under fume hood and vortex immediately for 1 to 2 seconds (proceed tube by tube to avoid incomplete lysis).

  7. Incubate for 10 minutes at between 18 and  25 °C. Protect from light.

  8. Add 250 μL of 1X PBS and vortex immediately for 1 to 2 seconds.

  9. Incubate for 10 minutes at between 18 and 25 °C. Protect from light.

  10. Sample is ready for acquisition.

OPTIONAL WASH STEP

  1. Proceed normally from step 1 to 9 and then add 3 mL of 1X PBS.

  2. Centrifuge for 5 minutes at 300 x g at room temperature.

  3. Aspirate the supernatant.

  4. Resuspend cells in 500  μL of PBS with 0.1% of formaldehyde.

  5. Sample is ready for acquisition.