Shreffler:Basophil Sensitization

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Contents

Overview

Materials

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. . pmid:15916720. PubMed HubMed [Hennersdorf]
  2. Knol EF, Mul FP, Jansen H, Calafat J, and Roos D. . pmid:1716273. PubMed HubMed [Knol]
  3. Shreffler WG. . pmid:16670519. PubMed HubMed [Shreffler]
All Medline abstracts: PubMed HubMed


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