Shreffler:Basophil Sensitization

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(Materials)
Current revision (10:48, 23 June 2010) (view source)
(Materials)
 
Line 17: Line 17:
*Dulbecco PBS cat nr 14190-094 lot...916276...
*Dulbecco PBS cat nr 14190-094 lot...916276...
*Lactic acid: 13,4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3,9 (BBlab)  
*Lactic acid: 13,4 mM lactate, 140 mM NaCl, 5 mM KCl, pH 3,9 (BBlab)  
-
*12% Tris (Merck 1.08382.0100)  
+
*12% Tris, pH 8.0 (Merck 1.08382.0100)  
*96U well plate Nunc 163320
*96U well plate Nunc 163320
*BSA Applicem A1391 →FLOW 0.5%BSA
*BSA Applicem A1391 →FLOW 0.5%BSA

Current revision

Contents

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:
    1. Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at 800xG –low brake (1).
    2. Discard upper layer (plasma supernatant) with vacuum suction or pipette.
    3. Transfer white interphase with mononuclear cells (PBMCs) from each leucosep tube into separate 14 ml PP Falcon tubes.
    4. Centrifuge tubes without any additional buffer, 800xG, 15 min, brake 3, 4 °C. 
From here on everything is kept on ice bath until sensitization.
    5. 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.
    6. 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) .
    7. Discard supernatant and place the pellet/PS tube on ice.
  3. IgE removal (keep all reagents on ice bath):
    1. Resuspend cell pellets in 3 mL lactic acid, put on ice for 5 min.
    2. Add 7 ml cold RPMI, 0.5% HSA, neutralize by adding 15 µl 12% Tris, centrifuge 600xG, 5min, brake 3, 4 °C.
    3. Discard supernatant and wash again with RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C
    4. Discard supernatant and resuspend to 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA. 

  4. Sensitization (here, all cells are sensitized with the same pool.):
    1. Transfer cell suspensions to Falcon 14 mL PP tubes.
    2. 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.)
    3. 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.
    4. Incubate for 1 hour at 37°C in incubator (5% CO2).
    5. Resupend cells carefully by pipetting and wash in ice cold RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C.
    6. Discard supernatant and wash in RPMI 0.5% HSA (RT) 600xG, 5min, brake 3, 4 °C.
    7. Discard supernatant and resuspend to 1/4 of initial blood volume (~20 ml) in RPMI 0.5% HSA (RT).
  5. 
Cell preparation: divide cells between two PP-tubes (+/- IL3) with 20 µl of IL3 (100 ng/ml) added/ml cell suspension for IL3 priming.
  6. Allergen preparation:
    1. Prepare in RPMI+0,5% HSA
      • r Der p2 10 µg/ml (10.000 ng/ml)
      • r Derp2 30ug/ml = 100ul+200ul buffer
    2. 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)
    3. Pipette 150 µl allergen/well to 96 –deepwell (2ml well) plate.
  7. Activation:
    1. Add 150 µl cells to existing 150 µl allergen/buffer in deep-well plate
    2. Incubate for 1 hour at 37°C in water bath/incubator (5% CO2)
  8. Stop reaction by adding 30 µl EDTA 200 mM to each well (final EDTA conc. ~20 mM)
  9. Centrifuge 800xG, 10min, brake 3, 4 °C
  10. 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

discuss this protocol

References

  1. Kleine Budde I, de Heer PG, van der Zee JS, and Aalberse RC. . pmid:11815734. PubMed HubMed [Kleine]

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