Shreffler:BioCSI Basophil Activation - Revision history

Wayne G. Shreffler: /* Procedure */

Wayne G. Shreffler — Wed, 16 Sep 2009 13:29:44 GMT

Procedure

←Older revision		Revision as of 13:29, 16 September 2009
Line 47:		Line 47:
	# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.		# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.
	# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.		# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.
-	#: These dilutions of Ag:Serum are now used 1:1 with washed cells in usual basophil activation protocol	+	#: '''These dilutions of Ag:Serum are now used 1:1 with washed cells in usual basophil activation''' [[Shreffler:Basophil Activation \| '''protocol''']]

	==Discussion==		==Discussion==

Wayne G. Shreffler: /* Procedure */

Wayne G. Shreffler — Wed, 16 Sep 2009 13:27:26 GMT

Procedure

←Older revision		Revision as of 13:27, 16 September 2009
Line 47:		Line 47:
	# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.		# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.
	# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.		# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.
		+	#: These dilutions of Ag:Serum are now used 1:1 with washed cells in usual basophil activation protocol

	==Discussion==		==Discussion==

Wayne G. Shreffler: /* Materials */

Wayne G. Shreffler — Wed, 24 Jun 2009 15:13:15 GMT

Materials

←Older revision		Revision as of 15:13, 24 June 2009
Line 11:		Line 11:
	* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)		* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
	* Staining Buffer (PBS + 2 mM EDTA + 0.5% BSA) (sterile filter, store at 4°C; aliquot in hood; expires in 2 months)		* 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)	+	* 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)
	* stimulant aliquots (pre-made, 30 μL aliquots, distributed by Mt. Sinai; stored at -20°C)		* stimulant aliquots (pre-made, 30 μL aliquots, distributed by Mt. Sinai; stored at -20°C)

Wayne G. Shreffler at 15:10, 24 June 2009

Wayne G. Shreffler — Wed, 24 Jun 2009 15:10:12 GMT

←Older revision		Revision as of 15:10, 24 June 2009
Line 15:		Line 15:

	----		----
		+	[[Image:biocsi_template_image.jpg]]

-		+	(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)

	==Procedure==		==Procedure==
Line 25:		Line 26:
	# Add 30 μL of RPMI to A1-A5 aliquots.		# Add 30 μL of RPMI to A1-A5 aliquots.
	# Set up a 96-well plate for serum and antigen dilutions.		# Set up a 96-well plate for serum and antigen dilutions.
-	~~[[Image:biocsi_template_image.jpg]]~~
-	~~(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)~~
	# Prepare serum/antigen dilutions as follows.		# Prepare serum/antigen dilutions as follows.
-	**Add 100 μL RPMI to S1, S2, and S3.	+	#:*Add 100 μL RPMI to S1, S2, and S3.
-	**Make serum dilution #1 by adding 100 μL serum to S1.	+	#:*Make serum dilution #1 by adding 100 μL serum to S1.
-	**Make successive 3 fold dilutions by transferring 50 μL from S1 to S2. Mix. Transfer 50 μL from S2 to S3 and mix.	+	#:*Make successive 3 fold dilutions by transferring 50 μL from S1 to S2. Mix. Transfer 50 μL from S2 to S3 and mix.
-	**Add 60 μL of S1 to A1 and A2. Vortex.	+	#:*Add 60 μL of S1 to A1 and A2. Vortex.
-	**Add 60 μL of S2 to A3. Vortex.	+	#:*Add 60 μL of S2 to A3. Vortex.
-	**Add 60 μL of S3 to A4. Vortex.	+	#:*Add 60 μL of S3 to A4. Vortex.
-	**Add 60 μL RPMI to A5. Vortex.	+	#:*Add 60 μL RPMI to A5. Vortex.
	# Incubate Eppendorf tubes A1-A5 and plate for 30 min at 37°C		# Incubate Eppendorf tubes A1-A5 and plate for 30 min at 37°C
	# Add 270 μL RPMI to wells B1-D5.		# Add 270 μL RPMI to wells B1-D5.
Line 40:		Line 39:
	# Add 0.6 μL anti-IgE to well 6 (2 μg/mL).		# Add 0.6 μL anti-IgE to well 6 (2 μg/mL).
	# Incubate 2.5 mL of RPMI to be used for diluting tubes A1-A5 after 30-minute incubation.		# Incubate 2.5 mL of RPMI to be used for diluting tubes A1-A5 after 30-minute incubation.
-	'''While samples are incubating…'''	+	#: '''While samples are incubating…'''
	# Spin whole blood from the green tip heparin tube at 300 x ''g'' for 10 min. Take off supernatant and fill with PBS to wash. Invert.		# Spin whole blood from the green tip heparin tube at 300 x ''g'' for 10 min. Take off supernatant and fill with PBS to wash. Invert.
	# Spin the sample again for 10 min at 300 x ''g''. Take off supernatant.		# Spin the sample again for 10 min at 300 x ''g''. Take off supernatant.
-	'''After antigen/serum incubation…'''	+	#: '''After antigen/serum incubation…'''
	# Add 480 μL RPMI to A1-A5 and vortex (use the 2.5 warm RPMI from step 11).		# Add 480 μL RPMI to A1-A5 and vortex (use the 2.5 warm RPMI from step 11).
	# Transfer 30 μL from “A” tubes to corresponding “B” tubes (A1 to B1, A2 to B2, etc.) with 50p multi-channel pipette. Mix.		# Transfer 30 μL from “A” tubes to corresponding “B” tubes (A1 to B1, A2 to B2, etc.) with 50p multi-channel pipette. Mix.

Wayne G. Shreffler: /* Procedure */

Wayne G. Shreffler — Wed, 24 Jun 2009 14:07:31 GMT

Procedure

←Older revision		Revision as of 14:07, 24 June 2009
Line 25:		Line 25:
	# Add 30 μL of RPMI to A1-A5 aliquots.		# Add 30 μL of RPMI to A1-A5 aliquots.
	# Set up a 96-well plate for serum and antigen dilutions.		# Set up a 96-well plate for serum and antigen dilutions.
-	[[Image:biocsi_template_image.~~pdf~~]]	+	[[Image:biocsi_template_image.jpg]]
	(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)		(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)
	# Prepare serum/antigen dilutions as follows.		# Prepare serum/antigen dilutions as follows.

Wayne G. Shreffler: /* Procedure */

Wayne G. Shreffler — Wed, 24 Jun 2009 14:05:01 GMT

Procedure

←Older revision		Revision as of 14:05, 24 June 2009
Line 25:		Line 25:
	# Add 30 μL of RPMI to A1-A5 aliquots.		# Add 30 μL of RPMI to A1-A5 aliquots.
	# Set up a 96-well plate for serum and antigen dilutions.		# Set up a 96-well plate for serum and antigen dilutions.
-	[[Image:biocsi_template_image.~~tiff~~]]	+	[[Image:biocsi_template_image.pdf]]
	(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)		(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)
	# Prepare serum/antigen dilutions as follows.		# Prepare serum/antigen dilutions as follows.
Line 48:		Line 48:
	# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.		# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.
	# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.		# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.
-
-

	==Discussion==		==Discussion==

Wayne G. Shreffler: 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...

Wayne G. Shreffler — Wed, 24 Jun 2009 14:03:16 GMT

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...

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 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.

This protocol is specifically for the BioCSI study.

==Materials==

* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)
* PBS + 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)
* 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)
* stimulant aliquots (pre-made, 30 μL aliquots, distributed by Mt. Sinai; stored at -20°C)

----

==Procedure==

# Obtain whole blood specimens collected in sodium heparin tube (green top).
# Record the total volume of blood in the wiki.
# Thaw 1 EW and 4 CR (30 ul) antigen aliquots. Label them A1-A5 (Ova=A1 and the CR aliquots=A2-A5).
# Add 30 μL of RPMI to A1-A5 aliquots.
# Set up a 96-well plate for serum and antigen dilutions.
[[Image:biocsi_template_image.tiff]]
(The green, blue, grey, and red colors represent 4 separate samples. Set up the plate for the number of samples received at one time.)
# Prepare serum/antigen dilutions as follows.
**Add 100 μL RPMI to S1, S2, and S3.
**Make serum dilution #1 by adding 100 μL serum to S1.
**Make successive 3 fold dilutions by transferring 50 μL from S1 to S2. Mix. Transfer 50 μL from S2 to S3 and mix.
**Add 60 μL of S1 to A1 and A2. Vortex.
**Add 60 μL of S2 to A3. Vortex.
**Add 60 μL of S3 to A4. Vortex.
**Add 60 μL RPMI to A5. Vortex.
# Incubate Eppendorf tubes A1-A5 and plate for 30 min at 37°C
# Add 270 μL RPMI to wells B1-D5.
# Add 300 μL RPMI to wells 6-8.
# Add 0.6 μL anti-IgE to well 6 (2 μg/mL).
# Incubate 2.5 mL of RPMI to be used for diluting tubes A1-A5 after 30-minute incubation.
'''While samples are incubating…'''
# Spin whole blood from the green tip heparin tube at 300 x ''g'' for 10 min. Take off supernatant and fill with PBS to wash. Invert.
# Spin the sample again for 10 min at 300 x ''g''. Take off supernatant.
'''After antigen/serum incubation…'''
# Add 480 μL RPMI to A1-A5 and vortex (use the 2.5 warm RPMI from step 11).
# Transfer 30 μL from “A” tubes to corresponding “B” tubes (A1 to B1, A2 to B2, etc.) with 50p multi-channel pipette. Mix.
# Transfer 30 μL from “B” tubes to corresponding “C” tubes and mix.
# Transfer 30 μL from “C” tubes to corresponding “D” tubes and mix.

==Discussion==
[[Talk:{{PAGENAME}}|discuss this protocol]]

==References==
<biblio>
#Hennersdorf pmid=15916720
#Knol pmid=1716273
#Shreffler pmid=16670519
</biblio>

==Contact==
*Who has experience with this protocol?
*[[Special:Emailuser/Wayne G. Shreffler|Email Wayne Shreffler through OpenWetWare]]

[[Category:ShreffLab]]

[[Category:Protocol]]

[[Category:Flow cytometry]]

[[Category:Immunology]]