Shreffler:Basophil Sensitization

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Overview

Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, a modification of method from Kleine.

  • Surface IgE on Basophils in PBMC preparations from non allergic individuals are removed by acid treatment and replaced with recombinant IgE.
  • Activation of the sensitized cells are measured with Basophile activation assay (BAT) by 1 h challenge with increasing concentrations of rec Derp2 and FACS monitoring of increased surface markers.

Materials

  • Vacuum VenoJect tubes with Na-heparin, Terumo, (Code: VT-100SH)
  • 
Centrifuge 1: P05-07-F408
  • Centrifuge 2: P05-07-F037
  • Incubator: P-05-07-F 482
  • BD FACS Lysing: cat nr 349202
  • BD FACS FLOW: cat nr 342003
  • Leucoseptubes, Greiner, Art. No: 227290.
  • Lymfoprep: Fresenius Kabi 1114547 lot 06106S0
  • Leucosep preparation: 15 ml lymfoprep in each leucosep, centrifuge 5 min 500xG
  • Dulbecco PBS cat nr 14190-094 lot...916276...
  • Lactic acid: 13,4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3,9 (BBlab)
  • 12% Tris (Merck 1.08382.0100)
  • 96U well plate Nunc 163320
  • BSA Applicem A1391 →FLOW 0.5%BSA
  • IL3 RD 203IL 10 µg/ml vials -20°C. Lot: 49000118324 GiL 711 s 87. →100 ng/ml in RPMI, HSA (1:100)
  • EDTA Sigma E6511 200 mM in PBS lot 868-14 010307
  • RPMI 1640 BE12-115F/U1 lot 6MB0203
  • HSA Sigma A1653 →RPMI 0.5 % /→PBS 0.5 %
  • Rec Derp2 HEK 30 µg/ml
  • Buffer preparations:
    • PBS+ 0.5% HSA A1653 cold
    • RPMI + 0.5% HSA A1653 cold/rt
  • Antibodies:
    • aCD63 FITC: A0507 lot 49…117695-2 2/2
    • aCD203c PE: A0224 lot 49…117695-1 1/2
    • aCD69 PerCP: A0028 lot 45…502063 1/2
    • aIgE Biotin: A0197 lot 72---036
    • SA-APC BD 554067 lot 49…132569

Procedure

Discussion

discuss this protocol

References

  1. Kleine Budde I, de Heer PG, van der Zee JS, and Aalberse RC. The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum. Int Arch Allergy Immunol. 2001 Dec;126(4):277-85. DOI:10.1159/000049524 | PubMed ID:11815734 | HubMed [Kleine]

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