Shreffler:Basophil Sensitization

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(Overview)
(Overview)
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==Overview==
==Overview==
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Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, modification of method from Kleine.   
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Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, a modification of method from Kleine.   
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*Surface IgE on Basophils in PBMC preparations from non allergic individuals are removed by acid treatment and replaced with recombinant IgE.  
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*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.
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�Procedure:
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Blood:
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Fresh drawn heparinised blood from non allergic donor: 80 ml.

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PBMC:
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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.
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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
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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.
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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)
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Discard supernatant and place the pellet/PS tube on ice.
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IgE removal:
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All reagents are kept on ice bath
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Cell pellet are resuspended in 3 ml Lactic acid.  
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On ice 5 min.
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Add 7 ml cold RPMI, 0.5% HSA.
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Neutralization by adding 15 µl 12% Tris
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Centrifuge 600xG, 5min, brake 3, 4 °C.
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Discard supernatant and wash additional  1x with RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C
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Discard supernatant and resuspend in 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA. 

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Sensitization:
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In PP Falcon 14 ml tubes.
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In this example all the cells (from 80 ml blood) are sensitized with the same pool.
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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)
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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
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Incubation for  1 hour at 37°C in incubator (5% CO2 )
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Resupend the cells carefully by pipetting and wash in ice cold RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C
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Discard supernatant and wash in RPMI 0.5% HSA (RT) 600xG, 5min, brake 3, 4 °C
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Discard supernatant and resuspend to 1/4 of initial blood volume (20 ml) in RPMI 0.5% HSA (RT)
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Cell preparation:
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The cells are split in two PP-tubes (+/- IL3) and 20 µl of IL3 (100 ng/ml) added/ml cell suspension for IL3 priming.
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Allergen preparation:
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Preparation in  RPMI+0,5% HSA
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r Der p2  10 µg/ml (10.000 ng/ml) 
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r Derp2  30ug/ml = 100ul+200ul buffer
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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)
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150 µl allergen/well in 96 –deepwell (2ml well) plate.
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Activation:
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150 µl cells + 150 µl allergen/buffer  in deep-well plate
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Incubation for  1 hour at 37°C in water bath/ incubator (5% CO2 )
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Reaction stopped by adding 30 µl EDTA 200 mM/well (final ~20 mM)
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Centrifugation 800xG, 10min, brake 3, 4 °C
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Supernatant removed and frozen for histamine determination
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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  
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==Materials==
==Materials==

Revision as of 11:18, 9 June 2010

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 (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

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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