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FLOWsurface with FACS FLOW 0.5% BSAand for FACS analysis like whole blood analysis. Basophils in each well 300 µl bloodas up to 50% lost the procedureand can be divided in two for staining with different antibody combinations.
Revision as of 13:02, 9 June 2010
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.
- 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
- Obtain 80 mL fresh drawn heparinised blood from non allergic donor.
- PBMC Isolation:
- Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at 800xG –low brake (1).
- Discard upper layer (plasma supernatant) with vacuum suction or pipette.
- Transfer white interphase with mononuclear cells (PBMCs) from each leucosep tube into separate 14 ml PP Falcon tubes.
- Centrifuge tubes without any additional buffer, 800xG, 15 min, brake 3, 4 °C. From here on everything is 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 transfer to a 10 ml polystyrene tube and wash one time with cold PBS, HSA 600xG, 5min, brake 3, 4 °C. (Note: with blood volumes >60 ml the cells are divided amongst several PS tubes) .
- Discard supernatant and place the pellet/PS tube on ice.
- IgE removal (keep all reagents on ice bath):
- Resuspend cell pellets in 3 mL lactic acid, put on ice for 5 min.
- Add 7 ml cold RPMI, 0.5% HSA, neutralize by adding 15 µl 12% Tris, centrifuge 600xG, 5min, brake 3, 4 °C.
- Discard supernatant and wash again with RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C
- Discard supernatant and resuspend to 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA.
- Sensitization (here, all cells are sensitized with the same pool.):
- Transfer cell suspensions to Falcon 14 mL PP tubes.
- To each cell suspension add 1 µl mixed pool of recAB consisting of 20% specific and 80% non specific IgE (Anti-Tox), 2 µg/ml (1µg/ml final conc.)
- If cells are sensitized with H12 and P4E, 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.
- Incubate for 1 hour at 37°C in incubator (5% CO2).
- Resupend 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: divide cells between two PP-tubes (+/- IL3) with 20 µl of IL3 (100 ng/ml) added/ml cell suspension for IL3 priming.
- Allergen preparation:
- Prepare in RPMI+0,5% HSA
- r Der p2 10 µg/ml (10.000 ng/ml)
- r Derp2 30ug/ml = 100ul+200ul buffer
- 10-fold dilutions: 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)
- Pipette 150 µl allergen/well to 96 –deepwell (2ml well) plate.
- Prepare in RPMI+0,5% HSA
- Add 150 µl cells to existing 150 µl allergen/buffer in deep-well plate
- Incubate for 1 hour at 37°C in water bath/incubator (5% CO2)
- Stop reaction by adding 30 µl EDTA 200 mM to each well (final EDTA conc. ~20 mM)
- Centrifuge 800xG, 10min, brake 3, 4 °C
- Remove supernatant and freeze for histamine determination.
FLOW: surface markers - Wash cell pellets with FACS FLOW 0.5% BSA; stain and prepare for FACS analysis like whole blood analysis. Basophils in each well correspond to 300 µl blood, as up to 50% of cells are lost throughout the procedure, and can be divided in two for staining with different antibody combinations.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare