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.

Analysis

Example Data in Kaluza Analysis File (download)

  1. Create an FSC-A vs. FSC-H dot plot and draw a region to encompass the singlets cell population and exclude the doublets.
  2. Create a FSC-A vs. SSC-A dot plot and apply the singlets gate onto this plot. Draw a region to encompass the Cells population. These are the singlets cell populations.
  3. Create a CD45-Krome Orange vs. SSC-A dot plot and apply the Cells gate onto this plot. Draw a region to encompass the CD45+ leukocytes. These are the singlet CD45+ Leukocytes cells.
  4. Create a CD3-PC7 vs. SSC-A dot plot and apply the Leukocytes gate onto the plot. Draw a region to exclude the CD3+ T cells and include the CD3- T cells + Lymphs (low SSC) + Monocytes (mid SSC) cell populations. Name this region the “Lymphs + monos (-CD3+ T cells)” gate.
  5. Create a CD294 (CRTH2) – Alexa Fluor 647 vs. CD203c-PE dot plot and apply the Lymphs + monos (- CD3+ T cells) gate onto this plot. Draw a region to encompass the CRTH2+ CD203c dim+ Cells.
  6. Create a CD63-Pacific Blue (PB) vs. CD203c-PE dot plot and apply the CRTH2+ CD203c dim+ Cells gate onto this plot. Select and apply a Quadrant gate onto this plot and adjust the quadrant lines to delineate the CRTH2+ CD63- CD203c dim+ resting basophils, CRTH2+ CD63+ CD203c bright+ activated basophils, CRTH2+ CD63+ CD203c- and CRTH2+ CD63- CD203c- cells.
  7. Record the desired statistics.