Shreffler:Basophil Sensitization: Difference between revisions

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(New page: ==Overview== Basophil activation in unfractionated samples such as PBMC (note basophils are less dense the Ficoll) or whole blood can be measured by changes in the expression of cell surf...)
 
 
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==Overview==
==Overview==
Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, a modification of method from Kleine. 


Basophil activation in unfractionated samples such as PBMC (note basophils are less dense the Ficoll) or whole blood can be measured by changes in the expression of cell surface markers or even intracellular events (e.g. phosphorylation, oxidative burst, calcium flux) by flow cytometry. Separate markers (e.g. as CD123+ HLA-DR-) are used to specifically identify basophils in addition to those used for assessment of activation.  
*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==
==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


* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
*Buffer preparations:
* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)
**PBS+ 0.5% HSA A1653 cold
* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
**RPMI + 0.5% HSA A1653 cold/rt
* Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)
* desired monoclonal antibodies (e.g. CD63-FITC, CD203c-PE, CD123 PE-Cy5, CD69-APC-Cy7, HLA DR-PE-CY7, CD3-APC, CD14-APC, CD19-APC, CD41a-APC)


Stimulants
*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.
#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.


'''May use other antigens for specific application. This will give an idea of the typical dose range we use for antigen-specific responses and controls.'''


Stimulant Preparation
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.
Basophil Medium (BM)= RPMI + 2 ng/mL human IL-3 (R&D, 203-IL-010/CF)
* Stock solution of IL-3 is 1,000X; (2 μg/mL aliquots stored in vials in -80°C); once vial is taken from -80°C, date, store 4°C and use within 1 month; vials stored in room 17-40
* For study, we need 2X stock (4 μM) in approximately 5 ml RPMI/subject
** 10 μL IL-3 in 5 mL RPMI
 
fMLP (Vendor, 47729-10MG-F) (Fisher, Nc9823482) – stored 4°C
* Stock solution at 4 mM in -30°C in 17-40
* We need 2X stock (2 μM) in 300 uL
** 1 μL of stock into 99 μL BM (100X) and vortex
* Take 15 μL of this intermediate dilution ad add to 285uL BM, vortex
 
Anti-IgE (Bethyl laboratories, A80-109A)
* Stock is 1 mg/mL (stored at 4°C in 17-40)
* We need 2 μg/mL (2X) in 300 μL
** 0.6 μL Anti-IgE in 300 uL BM
 
Peanut
* Stock is 15 mg/mL
* We need out top dilution to be 10<sup>7</sup> pg/mL=10 μg/mL
* Prepare 6 dilutions starting at 20 μg/mL and serially diluting 10-fold.
** Add 0.5 uL peanute extract to 375 μL BM and vortex (Tube PN1)
** Proceed to make 5 serial dilutions with 270 μL BM in each tube, diluting with 30 uL from each previous tube (Tubes PN2-PN6)
 
Egg White
* Stock is at 2 mg/mL
* Top dilution is 20 μg/mL
* Take 1μL EW stock and add to 99 μL BM and vortex
* Take 30 μL of this dilution and add to 270 μL BM (EW1)
* Serially dilute 10-fold 3 more times (EW 2-4) ie. 30μL of previous dilution in 270 μL BM
 
==Procedure==
Attach appropriate labels (study ID & condition) to polypropylene tubes.
 
  A. RPMI
  B. IL-3 (BM)
  C. FMLP
  D. PN1
  E. PN2
  F. PN3
  G. PN4
  H. PN5
  I. PN6
  J. Anti-IgE
  K. EW 1
  L. EW 2
  M. EW 3
  N. EW 4
# Make Stimulants (BM, IL-3, and fMLP, anti-IgE) according to instructions above.
# Transfer 250 μL of warm RPMI to tube A.
# Transfer 250 μL of each stimulant as above to polypropylene tubes B-N.
# Transfer 250 μL of patient whole blood to each tube A-N (total volume should now be 500 μL).
# Incubate for 30 minutes at 37°C (5% CO2) in an incubator, do not agitate!
# Prepare mAb staining cocktail.
# Add 50 μL cold (4°C) PBS w/ 20 mM EDTA to each tube to stop degranulation.
# Stain cells by adding 110 μL of the prepared Ab cocktail to tubes A-N.
# Incubate at 4°C for 30 minutes in the dark.
# Add 3 mL cold (4°C) staining buffer to each tube & centrifuge for 5 minutes @ 300x''g'' at 4°C.
# After spin, carefully aspirate supernatant to within 1/8’ of cells to leave pellet.
# Add 4 mL of 1X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O), dispense buffer from serological pipette into tube while vortexing at a low/medium speed.
# Incubate at room temperature in the dark for 15 minutes; may then place in refrigerator if leaving overnight.
# After incubation, spin tubes at 800 x ''g'' for 10 minutes.  (If incubated overnight, consider washing with 2 mL staining buffer.)
# Aspirate the supernatants carefully, then resuspend in 200 uL of staining buffer.
# Store at 4°C until ready to acquire.


==Discussion==
==Discussion==
Line 88: Line 79:
==References==
==References==
<biblio>
<biblio>
#Hennersdorf pmid=15916720
#Kleine pmid=11815734
#Knol pmid=1716273
#Shreffler pmid=16670519
</biblio>
</biblio>


==Contact==
==Contact==

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

  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. 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 | PubMed ID:11815734 | HubMed [Kleine]

Contact

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