Shreffler:Basophil Sensitization

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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,
+
Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, a modification of method from Kleine.  
-
Modification of method from Kleine  ADDIN REFMGR.CITE <Refman><Cite><Author>Kleine</Author><Year>2001</Year><RecNum>15</RecNum><IDText>The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>15</Ref_ID><Title_Primary>The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum</Title_Primary><Authors_Primary>Kleine,Budde,I</Authors_Primary><Authors_Primary>de Heer,P.G.</Authors_Primary><Authors_Primary>van der Zee,J.S.</Authors_Primary><Authors_Primary>Aalberse,R.C.</Authors_Primary><Date_Primary>2001/12</Date_Primary><Keywords>adverse effects</Keywords><Keywords>Allergens</Keywords><Keywords>analysis</Keywords><Keywords>Antibody Specificity</Keywords><Keywords>Basophils</Keywords><Keywords>Biological Assay</Keywords><Keywords>blood</Keywords><Keywords>diagnosis</Keywords><Keywords>Histamine Release</Keywords><Keywords>Human</Keywords><Keywords>Hypersensitivity,Immediate</Keywords><Keywords>Immunoglobulin E</Keywords><Keywords>immunology</Keywords><Keywords>methods</Keywords><Keywords>Radioallergosorbent Test</Keywords><Keywords>Support,Non-U.S.Gov&apos;t</Keywords><Reprint>Not in File</Reprint><Start_Page>277</Start_Page><End_Page>285</End_Page><Periodical>Int.Arch.Allergy Immunol.</Periodical><Volume>126</Volume><Issue>4</Issue><Address>Department of Immunopathology, CLB, Academic Medical Centre, University of Amsterdam, The Netherlands</Address><Web_URL>PM:11815734</Web_URL><ZZ_JournalStdAbbrev><f name="System">Int.Arch.Allergy Immunol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1)
+
-
Method:
+
*Surface IgE on Basophils in PBMC preparations from non allergic individuals are removed by acid treatment and replaced with recombinant IgE.  
-
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.
-
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
-
�Procedure:
+
*Antibodies:
 +
**aCD63 FITC: A0507 lot 49…117695-2 2/2
 +
**aCD203c PE-Cy7:
-
Blood:
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==Procedure==
-
Fresh drawn heparinised blood from non allergic donor: 80 ml.
  
+
#Obtain 80 mL fresh drawn heparinised blood from non allergic donor.

-
PBMC:
+
#PBMC Isolation:
-
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.  
+
##Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at  800xG –low brake (1).  
-
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 upper layer (plasma supernatant) with vacuum suction or pipette.  
-
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.
+
##Transfer white interphase with mononuclear cells (PBMCs) from each leucosep tube into separate 14 ml PP Falcon tubes.  
-
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)  
+
##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 place the pellet/PS tube on ice.
+
##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.
 +
#Activation:
 +
##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.
-
IgE removal:  
+
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.
-
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
+
-
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. 

+
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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)
+
-
 
+
-
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)
+
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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
+
-
 
+
-
 
+
-
 
+
-
ADDIN REFMGR.REFLIST Reference List
+
-
 
+
-
(1) Kleine B, I, de Heer PG, van der Zee JS, Aalberse RC. The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum. Int Arch Allergy Immunol 2001; 126(4):277-285.
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FILENAME #138359 v1 - Phl + mide krydssens + fb. PC og MH. Høj dose GiL08-003.  Side  PAGE 3/ NUMPAGES 4
+
-
 
+
-
==Materials==
+
-
Material:
+
-
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==
==Discussion==

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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