Shreffler:JAX Basophil Activation

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(Materials)
(Materials)
Line 16: Line 16:
* 5 mL round bottom polypropylene tubes (Falcon)
* 5 mL round bottom polypropylene tubes (Falcon)
* 1.5 mL eppendorf tubes
* 1.5 mL eppendorf tubes
-
* 15 mL conical tubes
+
* 15 and 50 mL conical tubes
* vacuum filter flasks (Corning #430186)
* vacuum filter flasks (Corning #430186)
* parafilm
* parafilm
* foil
* foil
* pipettes and tips
* pipettes and tips
 +
* serological pipettes (5, 10, 25)
==Procedure==
==Procedure==

Revision as of 15:47, 20 October 2009

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 the JAX study.

Materials

  • RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
  • IL-3 (R&D) 2 μg/μL
  • 1 X FACS lysing solution (made from 10X stock with dH2O; store at 4°C; expires in 1 week)
  • PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
  • 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, CD69-APC-Cy7, HLA DR-PE-Cy7, CD3-APC, CD41a-APC)
  • stimulant aliquots (pre-made, 30 μL aliquots, distributed by Shreffler Lab; stored at -20°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)

Procedure

  1. Obtain whole blood specimens collected in sodium heparin tube (green top).
  2. Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer. Label each stimulant and put aside to thaw.
  3. Record the total volume of blood on the data form.
  4. Aliquot 5 mL of RPMI into 15 mL conical tube and place it in a 37°C water bath to keep warm (for the dilution of stimulants and for the Unstained Control).
  5. Label eppendorf tubes for stimulants and 5 mL PP tubes for conditions (A-K).
  6. Prepare 5 (30 μL) aliquots of Basophil medium as follows:
    • Add 3.6 μL of IL-3 into 180 μL of warm RPMI
    • Aliquot 30 μL of RPMI + IL-3 into 5 (2 mL) eppendorf tubes labeled as Basophil medium 1 – Basophil medium 5.
    • Add 270 μL of warm RPMI into each tube.
  7. Add 270 μL of warm RPMI to the following stimulants: anti-IgE, Mouse allergen, and fMLP stimulants. Label the mouse tube as Mouse 1. Vortex all tubes for 5s.
  8. Add 135 μL of warm RPMI to the Calcium tube and the Ionophore tube. Transfer both diluted stimulants into a single 2mL eppendorf tube labeled Ca/I. Vortex
  9. Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
    • Label four eppendorf tubes as Mouse 2 – Mouse 5.
    • Transfer 270 μL of the Basophil medium prepared above to each of the Mouse 2-5 tubes.
    • Transfer 30 μL from Mouse 1 stimulant into Mouse 2 tube, vortex. Repeat this from Mouse 2 tube into Mouse 3 tube and continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 –Mouse 5). Vortex after each dilution.
    • Note: if processing more than one sample, take care to track participant ID.
    Image:jax_stim.tiff
  10. Transfer 250 μL of warm RPMI into tubes A and B.
  11. Add 250 μL of each stimulant to the appropriate polypropylene tube (C-K) according to the table above.
  12. IN THE HOOD, add 250 μL of patient blood to all eleven tubes.
  13. Incubate tubes for 20 minutes in 37°C incubator (5% CO2). Do not shake tubes!
  14. While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
    • To 1.2 mL staining buffer, add 90 μL each of the CD63, CD203c, CD123, CD3 and CD41a and 45 μL each of the HLA-DR and CD69 mAbs.
  15. After 20 minutes of incubation, remove the samples and add 50 μL cold PBS w/ 20mM EDTA.
  16. Add 110 μL of antibody cocktail to tubes B -> K (DO NOT ADD TO A!).
  17. Incubate for 30 min at 4°C. Protect from light.
  18. Add 4 mL of 1x FACS lysing solution. Cover with parafilm and invert repeatedly until pellet is fully resuspended.
  19. Place tubes in the dark for 15 minutes or overnight in the refrigerator.
  20. Centrifuge for 5 minutes at 800 x g. Decant supernatant and resuspend pellet in 100 μL staining buffer.
  21. Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
  22. Ship to Shreffler Lab at room temperature.

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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