Shreffler:Cisplatin Allergen

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Contents

Overview

Basophil activation in unfractionated samples such as PBMC (note basophils are less dense than 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. as CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation.

This protocol is specifically for our collaboration with Aleena Banerji in Allergy Associates and Oncology.

Materials

  • RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
  • IL-3 (R&D) 2 μg/mL
  • 1 X FACS lysing solution (made from 10X stock with dH2O; store at 4°C; expires in 1 month)
  • PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 month)
  • Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
  • monoclonal antibodies (e.g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, HLA DR-PE-Cy7)
  • stimulant aliquots (pre-made, 30 μL aliquots, distributed by Shreffler Lab; stored at -80°C)
  • 5 mL round bottom polypropylene tubes (Falcon)
  • 1.5 mL eppendorf tubes
  • 15 and 50 mL conical tubes
  • vacuum filter flasks (Corning #430186)
  • parafilm
  • foil
  • pipettes and tips
  • serological pipettes (5, 10, 25)
  • Platin drug (20X concentration)

Procedure

  1. Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
  2. Remove pre-made stimulants (fMLP, Drug, Anti-IgE) from freezer.
  3. Label each stimulant and put aside to thaw.
  4. Aliquot 5 mL of RPMI into 15 mL conical tube
  5. Place RPMI in a 37°C water bath for a minimum of 10 minutes.
  6. Label four eppendorf tubes for Drug 2-5, and nine 5 mL PP tubes for conditions (A-I).
  7. Prepare Basophil medium (BM) as follows (set aside for step 10):
    • Add 4 μL of IL-3 into 2.0 mL of warm RPMI (use 5 mL PP tube). Vortex
  8. Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP, Drug 1.
  9. Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:
    • Add 270 μL of prepared Basophil medium to Drug 1 stimulant. Vortex
    • Transfer 270 μL of the Basophil medium prepared above to each of the eppendorf tubes labeled Drug 2-5.
    • Transfer 30 μL from Drug 1 stimulant into Drug 2 eppendorf, vortex. Repeat this from Drug 2 eppendorf into Drug 3 eppendorf
    • Continue making 10-fold dilutions in the same manner until all four Drug dilutions have been prepared (Drug 2 –Drug 5). Vortex after each dilution.
  10. Transfer 250 μL of warm RPMI into tubes A and B.
  11. Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (drug allergen for tube 'G'; basophil medium for tube 'C', etc.).
  12. Gently mix blood by inverting green top tube 4 times.
  13. IN THE HOOD, add 250 μL of patient blood to all eleven tubes.
  14. Incubate tubes for 20 minutes in 37°C incubator (5% CO2). Do not shake tubes!
  15. While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
    • To 900 uL staining buffer, add 90 μL each of:
    • CD63
    • CD203c
    • CD123
    and 12 μL of:
    • HLA-DR
    Vortex
  16. After the 20 minutes of incubation, remove the samples.
  17. Put samples on ice.
  18. Add 110 μL of antibody cocktail to tubes B -> K (DO NOT ADD TO A!).
  19. Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
  20. Incubate for 20 min at 4°C. Protect from light.
  21. Add 2 mL staining buffer.
  22. Invert tubes 3X to mix
  23. Centrifuge for 5 minutes at 300g, RT
  24. Aspirate supernatant, taking care not to disturb cell pellet.
  25. Add 3 mL of 1x FACS lysing solution one tube at a time, recap and invert repeatedly until pellet is fully resuspended.
  26. Place tubes in the dark for 15 minutes or overnight in the refrigerator.
  27. Top off tube with staining buffer
  28. Centrifuge for 5 minutes at 800g, RT.
  29. Decant supernatant (do not double dump).
  30. Resuspend pellet in 50 μL staining buffer.
  31. Aquire

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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