Shreffler:Basophil Sensitization: Difference between revisions
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==Overview== | ==Overview== | ||
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. | ||
*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== | ==Materials== | ||
Line 96: | 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 | ||
Line 111: | Line 32: | ||
*Antibodies: | *Antibodies: | ||
**aCD63 FITC: A0507 lot 49…117695-2 2/2 | **aCD63 FITC: A0507 lot 49…117695-2 2/2 | ||
**aCD203c PE | **aCD203c PE-Cy7: | ||
==Procedure== | ==Procedure== | ||
#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. | |||
#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. | |||
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== | ==Discussion== |
Latest revision as of 07:48, 23 June 2010
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
- 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
- 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.
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
References
- Kleine Budde I, de Heer PG, van der Zee JS, and 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 Dec;126(4):277-85. DOI:10.1159/000049524 |
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare