Shreffler:Basophil Sensitization: Difference between revisions
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==Overview== | ==Overview== | ||
Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, modification of method from Kleine. | 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. | |||
IgE | |||
==Materials== | ==Materials== |
Revision as of 09:18, 9 June 2010
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
References
- 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 |
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare