Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, modification of method from Kleine.
Blood: Fresh drawn heparinised blood from non allergic donor: 80 ml. PBMC: Fill the blood in lymfoprep prepared leucosep tubes (maximum 20 ml in each). Centrifuge 20 min at 800xG –low brake (1). Discard upper layer -plasma supernatant with vacuum suction or pipette. The white interphase with mononuclear cells, PBMC from one leucosep is poured to one 14 ml PP Falcon tube. Centrifuge without any additional buffer, 800xG, 15 min, brake 3, 4 °C. From here everything are kept on ice bath until sensitization Discard supernatant and resuspend each pellet in cold PBS, HSA. Pool the cells and wash with 10 ml of the same buffer, 600xG, 10 min, brake 3, 4 °C. Discard supernatant, resuspend cell pellet in cold PBS, HSA and move to 10 ml polystyrene tube and wash additional 1x with cold PBS, HSA 600xG, 5min, brake 3, 4 °C. (Note: with blood volumes >60 ml the cells are divided in more PS tubes) Discard supernatant and place the pellet/PS tube on ice.
IgE removal: All reagents are kept on ice bath
Cell pellet are resuspended in 3 ml Lactic acid. On ice 5 min. Add 7 ml cold RPMI, 0.5% HSA. Neutralization by adding 15 µl 12% Tris Centrifuge 600xG, 5min, brake 3, 4 °C. Discard supernatant and wash additional 1x with RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C Discard supernatant and resuspend in 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA. Sensitization: In PP Falcon 14 ml tubes.
In this example all the cells (from 80 ml blood) are sensitized with the same pool.
To each µl cell suspension we add 1 µl mixed pool of recAB . The pool consist of 20 % specific and 80 % non specifik IgE ( Anti Tox), 2 µg/ml ( 1µg/ml final sens)
If cells are sensitized with H12 and P4E we use a pool with 0.2 µg/ml H12 + 0.2 µg/ml H10 + 1.6 µg/ml a-Tox and add 800 µl of this pool to the total volume of 800 µl cells
Incubation for 1 hour at 37°C in incubator (5% CO2 ) Resupend the cells carefully by pipetting and wash in ice cold RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C Discard supernatant and wash in RPMI 0.5% HSA (RT) 600xG, 5min, brake 3, 4 °C Discard supernatant and resuspend to 1/4 of initial blood volume (20 ml) in RPMI 0.5% HSA (RT) Cell preparation: The cells are split in two PP-tubes (+/- IL3) and 20 µl of IL3 (100 ng/ml) added/ml cell suspension for IL3 priming.
Allergen preparation: Preparation in RPMI+0,5% HSA
r Der p2 10 µg/ml (10.000 ng/ml) r Derp2 30ug/ml = 100ul+200ul buffer
Further 10 fold dilution: 50 µl+ 450 µl buffer to 10.000-1000-100-10-1-0,1-0,01-0,001 ng/ml (final activation 5000-0,0005 ng/ml)
150 µl allergen/well in 96 –deepwell (2ml well) plate.
Activation: 150 µl cells + 150 µl allergen/buffer in deep-well plate Incubation for 1 hour at 37°C in water bath/ incubator (5% CO2 ) Reaction stopped by adding 30 µl EDTA 200 mM/well (final ~20 mM) Centrifugation 800xG, 10min, brake 3, 4 °C Supernatant removed and frozen for histamine determination
FLOW, surface markers Cell pellet are washed with FACS FLOW 0.5 % BSA, stained and prepared for FACS analysis like whole blood analysis. Basophils in each well corresponds 300 µl blood as up to 50% is usually lost during the whole procedure and can be divided in two for staining with different antibody combinations
- 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
- 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
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare