Shreffler:PM Basophil Activation

Overview

Basophil activation in unfractionated samples, such as PBMC (note: basophils are less dense the Ficoll) or whole blood, can be measured by changes in the expression of cell surface markers or even intracellular events (e.g. phosphorylation, oxidative burst, calcium flux) by flow cytometry. Separate markers (e.g. CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation.

Materials

1. RPMI medium (cellgro/Mediatech, 10-040-CV)
   • Store at 4° in dark
2. 1 X FACS lysing solution (BD Biosciences)
   • Make from 10X stock with dH2O; store at 4°C; expires in 1 week
3. PBS + 20 mM EDTA
   • Sterile filter, store at 4°C; expires in 1 week
4. Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA)
   • Sterile filter, store at 4°C; aliquot in hood; expires in 2 months
5. Desired monoclonal antibodies
   • CD63-FITC (30 μL, BD 557288)
   • CD203c-PE (30 μL, Beckman Coulter IM3575)
   • CD123 PE-Cy5 (30 μL, BD 551065)
   • CD69-APC-Cy7 (15 μL, BD 557756)
   • HLA DR-PE-CY7 (15 μL, BD 335795)
6. Stimulants
   • Anti-IgE (Bethyl laboratories A80-109A) - store at 4°C
     • 0.6uL stock (stock: 1 mg/mL) in 300 uL RPMI
   • fMLP (Sigma Alderich 47729-10MG-F)
     • Stock solution at 4 mM in -30°C
     • We need to prepare 2X solution (2 μM) in 300 uL
       • 1 μL of stock into 99 μL RPMI and vortex
       • Take 15 μL of this intermediate dilution ad add to 285uL RPMI, vortex

Procedure

Attach appropriate labels (study ID & condition) to polypropylene tubes.

• A. RPMI
• B. FMLP
• C. Anti-IgE

1. Make Stimulants (RPMI, fMLP, anti-IgE) according to instructions above.
2. Transfer 250 μL of warm RPMI to tube A.
3. Transfer 250 μL of each stimulant as above to polypropylene tubes B-C.
4. Transfer 250 μL of patient whole blood to each tube A-C (total volume should now be 500 μL).
5. Incubate for 15 minutes at 37°C (5% CO2) in an incubator. Do not agitate!
6. Prepare mAb staining cocktail by mixing indicated volumes of each of the mAbs (above) into 350 μL of staining buffer. Vortex.
7. Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
8. Centrifuge samples at 300g for 5 minutes.
9. Pipette and store 200 uL of supernatant for histamine.
10. Resuspend cells in 200 uL of cold staining buffer.
11. Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-C.
12. Incubate at 4°C for 30 minutes in the dark.
13. Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300g at 4°C.
14. After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
15. Add 4 mL of 1X FACS lysing solution, dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
16. Incubate at room temperature in the dark for 15 minutes.
17. After incubation, spin tubes at 800g for 10 minutes.
18. Aspirate the supernatants carefully, then resuspend in 130 uL of staining buffer and transfer to eppendorf tubes.
19. Seal with parafilm and ship to MGH for acquisition.

References

1. Hennersdorf F, Florian S, Jakob A, Baumgärtner K, Sonneck K, Nordheim A, Biedermann T, Valent P, and Bühring HJ. Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation. Cell Res. 2005 May;15(5):325-35. DOI:10.1038/sj.cr.7290301 |
2. Knol EF, Mul FP, Jansen H, Calafat J, and Roos D. Monitoring human basophil activation via CD63 monoclonal antibody 435. J Allergy Clin Immunol. 1991 Sep;88(3 Pt 1):328-38. DOI:10.1016/0091-6749(91)90094-5 |
3. Shreffler WG. Evaluation of basophil activation in food allergy: present and future applications. Curr Opin Allergy Clin Immunol. 2006 Jun;6(3):226-33. DOI:10.1097/01.all.0000225165.83144.2f |