Shreffler:Basophil Sensitization

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, pH 8.0 (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-Cy7:

Procedure

 * 1) Obtain 80 mL fresh drawn heparinised blood from non allergic donor.
 * 2) PBMC Isolation:
 * 3) Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at 800xG –low brake (1).
 * 4) Discard upper layer (plasma supernatant) with vacuum suction or pipette.
 * 5) Transfer white interphase with mononuclear cells (PBMCs) from each leucosep tube into separate 14 ml PP Falcon tubes.
 * 6) Centrifuge tubes without any additional buffer, 800xG, 15 min, brake 3, 4 °C.  From here on everything is kept on ice bath until sensitization.
 * 7) 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.
 * 8) 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).
 * 9) Discard supernatant and place the pellet/PS tube on ice.
 * 10) IgE removal (keep all reagents on ice bath):
 * 11) Resuspend cell pellets in 3 mL lactic acid, put on ice for 5 min.
 * 12) Add 7 ml cold RPMI, 0.5% HSA, neutralize by adding 15 µl 12% Tris, centrifuge 600xG, 5min, brake 3, 4 °C.
 * 13) Discard supernatant and wash again with RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C
 * 14) Discard supernatant and resuspend to 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA.
 * 15) Sensitization (here, all cells are sensitized with the same pool.):
 * 16) Transfer cell suspensions to Falcon 14 mL PP tubes.
 * 17) 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.)
 * 18) 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.
 * 19) Incubate for 1 hour at 37°C in incubator (5% CO2).
 * 20) Resupend cells carefully by pipetting and wash in ice cold RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C.
 * 21) Discard supernatant and wash in RPMI 0.5% HSA (RT) 600xG, 5min, brake 3, 4 °C.
 * 22) Discard supernatant and resuspend to 1/4 of initial blood volume (~20 ml) in RPMI 0.5% HSA (RT).
 * 23)  Cell preparation: divide cells between two PP-tubes (+/- IL3) with 20 µl of IL3 (100 ng/ml) added/ml cell suspension for IL3 priming.
 * 24) Allergen preparation:
 * 25) Prepare in RPMI+0,5% HSA
 * 26) *r Der p2 10 µg/ml (10.000 ng/ml)
 * 27) *r Derp2 30ug/ml = 100ul+200ul buffer
 * 28) 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)
 * 29) Pipette 150 µl allergen/well to 96 –deepwell (2ml well) plate.
 * 30) Activation:
 * 31) Add 150 µl cells to existing 150 µl allergen/buffer in deep-well plate
 * 32) Incubate for 1 hour at 37°C in water bath/incubator (5% CO2)
 * 33) Stop reaction by adding 30 µl EDTA 200 mM to each well (final EDTA conc. ~20 mM)
 * 34) Centrifuge 800xG, 10min, brake 3, 4 °C
 * 35) 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.

Discussion
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Contact

 * Who has experience with this protocol?
 * Email Wayne Shreffler through OpenWetWare