Shreffler:Basophil Sensitization

Procedure

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

 A. RPMI
 B. IL-3 (BM)
 C. FMLP
 D. PN1
 E. PN2
 F. PN3
 G. PN4
 H. PN5
 I. PN6
 J. Anti-IgE
 K. EW 1
 L. EW 2
 M. EW 3
 N. EW 4

1. Make Stimulants (BM, IL-3, and 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-N.
4. Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
5. Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
6. Prepare mAb staining cocktail.
7. Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
8. Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
9. Incubate at 4°C for 30 minutes in the dark.
10. Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300xg at 4°C.
11. After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
12. Add 4 mL of 1X FACS lysing solution (made from 10X stock with dH₂O), dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
13. Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
14. After incubation, spin tubes at 800 x g for 10 minutes. (If incubated overnight, consider washing with 2 mL staining buffer.)
15. Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
16. Store at 4°C until ready to acquire.

Discussion

discuss this protocol

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 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare