Shreffler:JAX 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. 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)
• 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 Mt. Sinai; stored at -20°C)

Procedure

1. Obtain whole blood specimens collected in sodium heparin tube (green top).
2. Record the total volume of blood on the data form.
3. 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).
4. Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer. Label each stimulant and put aside to thaw.
5. 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.
6. Add 270 μL of warm RPMI to the following stimulants: anti-IgE, Mouse allergen, and fMLP stimulants. Label the mouse tube as Mouse1. Vortex all tubes for 5s.
7. 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
8. Prepare 10-fold serial dilutions of the Mouse1 stimulant as follows:
   • Label four eppendorf tubes as Mouse2 – Mouse5.
   • Transfer 270 μL of the Basophil medium prepared above to each of the Mouse2-5 tubes.
   • Transfer 30 μL from Mouse1 stimulant into Mouse2 tube, vortex. Repeat this from Mouse2 tube into Mouse3 tube and continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse2 –Mouse5). Vortex after each dilution.

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