Shreffler:Basophil Sensitization - Revision history

Wayne G. Shreffler: /* Materials */

Wayne G. Shreffler — Wed, 23 Jun 2010 14:48:02 GMT

Materials

←Older revision		Revision as of 14:48, 23 June 2010
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

Wayne G. Shreffler: /* Materials */

Wayne G. Shreffler — Fri, 11 Jun 2010 20:47:20 GMT

Materials

←Older revision		Revision as of 20:47, 11 June 2010
Line 32:		Line 32:
	*Antibodies:		*Antibodies:
	**aCD63 FITC: A0507 lot 49…117695-2 2/2		**aCD63 FITC: A0507 lot 49…117695-2 2/2
-	**aCD203c PE~~: A0224 lot 49…117695~~-~~1 1/2~~	+	**aCD203c PE-Cy7:
-	**aCD69 PerCP: ~~A0028 lot 45…502063 1/2~~	+
-	**aIgE Biotin: A0197 lot 72---036	+
-	**SA-APC BD 554067 lot 49…132569	+

	==Procedure==		==Procedure==

Alex Ma: /* Procedure */

Alex Ma — Wed, 09 Jun 2010 17:02:42 GMT

Procedure

←Older revision		Revision as of 17:02, 9 June 2010
Line 75:		Line 75:


-	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.	+	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==

Alex Ma: /* Procedure */

Alex Ma — Wed, 09 Jun 2010 17:00:09 GMT

Procedure

←Older revision		Revision as of 17:00, 9 June 2010
Line 65:		Line 65:
	##*r Der p2 10 µg/ml (10.000 ng/ml)		##*r Der p2 10 µg/ml (10.000 ng/ml)
	##*r Derp2 30ug/ml = 100ul+200ul buffer		##*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.

-	~~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.~~	+	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.
-		+
-	~~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)~~	+
-	~~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 	+

	==Discussion==		==Discussion==

Alex Ma: /* Procedure */

Alex Ma — Wed, 09 Jun 2010 16:55:11 GMT

Procedure

←Older revision		Revision as of 16:55, 9 June 2010
Line 52:		Line 52:
	##Discard supernatant and wash again with RPMI 0.5% HSA 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.		##Discard supernatant and resuspend to 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA.
-	#Sensitization:	+	#Sensitization (here, all cells are sensitized with the same pool.):
-	~~In PP Falcon 14 ml tubes.~~	+	##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.)
-	~~In this example~~ all ~~the~~ cells ~~(from 80 ml blood)~~ are sensitized with the same pool.	+	##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).
-	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~~)	+	##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.
-	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	+	##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.
-	~~Incubation~~ for 1 hour at 37°C in incubator (5% CO2 )	+	#Allergen preparation:
-	Resupend ~~the~~ cells carefully by pipetting and wash in ice cold RPMI 0.5% HSA 600xG, 5min, brake 3, 4 °C	+	##Prepare in RPMI+0,5% HSA
-	Discard supernatant and wash in RPMI 0.5% HSA (RT) 600xG, 5min, brake 3, 4 °C	+	##*r Der p2 10 µg/ml (10.000 ng/ml)
-	Discard supernatant and resuspend to 1/4 of initial blood volume (20 ml) in RPMI 0.5% HSA (RT)	+	##*r Derp2 30ug/ml = 100ul+200ul buffer
-	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)		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)

Alex Ma: /* Procedure */

Alex Ma — Wed, 09 Jun 2010 16:45:08 GMT

Procedure

←Older revision		Revision as of 16:45, 9 June 2010
Line 38:		Line 38:

	==Procedure==		==Procedure==
-	#Obtain 80 mL fresh drawn heparinised blood from non allergic donor.	+	#Obtain 80 mL fresh drawn heparinised blood from non allergic donor.
-		+
	#PBMC Isolation:		#PBMC Isolation:
	##Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at 800xG –low brake (1).		##Fill lymfoprep prepared leucosep tubes with blood (maximum 20 ml in each), centrifuge 20 min at 800xG –low brake (1).
Line 48:		Line 47:
	##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, 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.		##Discard supernatant and place the pellet/PS tube on ice.
-
	#IgE removal (keep all reagents on ice bath):		#IgE removal (keep all reagents on ice bath):
	##Resuspend cell pellets in 3 mL lactic acid, put on ice for 5 min.		##Resuspend cell pellets in 3 mL lactic acid, put on ice for 5 min.
Line 54:		Line 52:
	##Discard supernatant and wash again with RPMI 0.5% HSA 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.		##Discard supernatant and resuspend to 1/100 (0.8 ml) of the original blood volume in RPMI 0.5%HSA.
-
	#Sensitization:		#Sensitization:
	In PP Falcon 14 ml tubes.		In PP Falcon 14 ml tubes.

Alex Ma: /* Procedure */

Alex Ma — Wed, 09 Jun 2010 16:41:21 GMT

Procedure

←Older revision		Revision as of 16:41, 9 June 2010
Line 38:		Line 38:

	==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:
		+	In PP Falcon 14 ml tubes.
		+
		+	In this example all the cells (from 80 ml blood) are sensitized with the same pool.
		+
		+	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)
		+	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 

	==Discussion==		==Discussion==

Alex Ma: /* Overview */

Alex Ma — Wed, 09 Jun 2010 16:18:49 GMT

Overview

←Older revision		Revision as of 16:18, 9 June 2010
Line 1:		Line 1:
	==Overview==		==Overview==
-	Protocol for stripping IgE from basophils, re-sensitize with serum IgE and allergen specific basophile activation, modification of method from Kleine.	+	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.
-		+
-		+
-	~~�Procedure:~~	+
-		+
-	~~Blood:~~	+
-	~~Fresh drawn heparinised blood~~ from non allergic ~~donor: 80 ml.~~	+
-	~~PBMC:~~	+
-	~~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.~~	+
-	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 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 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)	+
-	~~Discard supernatant and place the pellet/PS tube on ice.~~	+
-		+
-		+
-	IgE ~~removal:~~	+
-	~~All reagents are kept on ice bath~~	+
-		+
-	~~Cell pellet are resuspended in 3 ml Lactic acid~~.	+
-	~~On ice 5 min.~~	+
-	~~Add 7 ml cold RPMI, 0.5% HSA.~~	+
-	~~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.~~	+
-	~~Sensitization:~~	+
-	~~In PP Falcon 14 ml tubes.~~	+
-		+
-	~~In this example all the cells (from 80 ml blood) are~~ sensitized ~~with the same pool.~~	+
-		+
-	~~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)~~	+
-	~~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 	+

	==Materials==		==Materials==

Alex Ma: /* Overview */

Alex Ma — Wed, 09 Jun 2010 16:15:13 GMT

Overview

←Older revision		Revision as of 16:15, 9 June 2010
Line 1:		Line 1:
	==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, modification of method from Kleine.
-	~~Modification~~ of method from Kleine	+

-	~~Method:~~
-	~~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.~~

Alex Ma: /* Overview */

Alex Ma — Wed, 09 Jun 2010 16:12:32 GMT

Overview

←Older revision		Revision as of 16:12, 9 June 2010
Line 1:		Line 1:
	==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,
-	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'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)	+	Modification of method from Kleine

	Method:		Method:
Line 66:		Line 66:

	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 		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==		==Materials==