Shreffler:CFSE Labeling - Revision history

Wayne G. Shreffler: Undo revision 313541 by Wayne G. Shreffler (Talk)

Wayne G. Shreffler — Wed, 10 Jun 2009 18:23:55 GMT

Undo revision 313541 by Wayne G. Shreffler (Talk)

←Older revision		Revision as of 18:23, 10 June 2009
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	==Overview==		==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~~.	+	CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.

	==Materials==		==Materials==

-	* ~~RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)~~	+	*15 mL polypropylene tubes
-	* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)	+	*[[PBS]]
-	* PBS ~~+ 20 mM EDTA (sterile filter, store at 4°C; expires in 1 week)~~	+	*5,6-CFDA/SE [http://products.invitrogen.com:80/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=C1157&messageType=catProductDetail Invitrogen C1157] dissolved in DMSO at 5 mM and stored at -20°C.
-	* ~~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~~==	+
-	~~'''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~~.~~'''~~	+
-		+

	==Procedure==		==Procedure==

Wayne G. Shreffler at 18:21, 10 June 2009

Wayne G. Shreffler — Wed, 10 Jun 2009 18:21:53 GMT

←Older revision		Revision as of 18:21, 10 June 2009
Line 1:		Line 1:
	==Overview==		==Overview==

-	~~CFSE is used to fluorescently label live cells~~ in ~~order to track them. 5,6-CFDA/SE is membrane permeable, but trapped~~ in the cell ~~when converted by~~ intracellular ~~esterases~~. ~~CFSE is equally partitioned to daughter cells during division and can be~~ used to ~~measure cell proliferation~~.	+	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.

	==Materials==		==Materials==

-	*~~15 mL polypropylene tubes~~	+	* RPMI medium (cellgro, 10-040-CV; store at 4°C in dark)
-	*[[PBS]]	+	* 1 X FACS lysing solution (made from 10X stock with dH<sub>2</sub>O; store at 4°C; expires in 1 week)
-	*5,~~6-CFDA/SE [http://products~~.~~invitrogen~~.~~com:80/ivgn/en/US/adirect/invitrogen?cmd~~=~~catProductDetail&entryPoint~~=~~adirect&productID~~=~~C1157&messageType~~=~~catProductDetail Invitrogen C1157] dissolved in DMSO at 5 mM and stored at~~ -~~20°C~~.	+	* 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)
		+
		+	==Stimulants==
		+	'''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.'''
		+

	==Procedure==		==Procedure==

Kehinde Kassim at 21:06, 3 June 2009

Kehinde Kassim — Wed, 03 Jun 2009 21:06:27 GMT

←Older revision		Revision as of 21:06, 3 June 2009
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	[[Category:Protocol]]		[[Category:Protocol]]

-	[[Category:Flow ~~Cytometry~~]]	+	[[Category:Flow cytometry]]

	[[Category:Immunology]]		[[Category:Immunology]]

Kehinde Kassim at 21:05, 3 June 2009

Kehinde Kassim — Wed, 03 Jun 2009 21:05:24 GMT

←Older revision		Revision as of 21:05, 3 June 2009
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	[[Category:Protocol]]		[[Category:Protocol]]
		+
		+	[[Category:Flow Cytometry]]

	[[Category:Immunology]]		[[Category:Immunology]]

Wayne G. Shreffler at 01:10, 28 May 2009

Wayne G. Shreffler — Thu, 28 May 2009 01:10:09 GMT

←Older revision		Revision as of 01:10, 28 May 2009
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	*[[Special:Emailuser/Wayne G. Shreffler\|Email Wayne Shreffler through OpenWetWare]]		*[[Special:Emailuser/Wayne G. Shreffler\|Email Wayne Shreffler through OpenWetWare]]

		+	[[Category:ShreffLab]]

	[[Category:Protocol]]		[[Category:Protocol]]

	[[Category:Immunology]]		[[Category:Immunology]]

Wayne G. Shreffler: /* Contact */

Wayne G. Shreffler — Thu, 21 May 2009 14:47:08 GMT

Contact

←Older revision		Revision as of 14:47, 21 May 2009
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	[[Category:Protocol]]		[[Category:Protocol]]

-	[[~~Cateogory~~:Immunology]]	+	[[Category:Immunology]]

Wayne G. Shreffler: /* Contact */

Wayne G. Shreffler — Thu, 21 May 2009 14:41:42 GMT

Contact

←Older revision		Revision as of 14:41, 21 May 2009
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	[[Category:Protocol]]		[[Category:Protocol]]
		+
	[[Cateogory:Immunology]]		[[Cateogory:Immunology]]

Wayne G. Shreffler at 14:41, 21 May 2009

Wayne G. Shreffler — Thu, 21 May 2009 14:41:14 GMT

←Older revision		Revision as of 14:41, 21 May 2009
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	#Lyons pmid=8176234		#Lyons pmid=8176234
	</biblio>		</biblio>
-	~~[[Category:Protocol]]~~	+

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

Wayne G. Shreffler: /* Procedure */

Wayne G. Shreffler — Thu, 21 May 2009 14:39:42 GMT

Procedure

←Older revision		Revision as of 14:39, 21 May 2009
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	#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.		#Suspend PBMCs at 10x10<sup>6</sup> cells/mL in PBS alone.
-	#~~Cells should be in PBS for~~ CFSE ~~labeling~~.	+	#Ensure that cells are uniformly suspended when CFSE is added.
	#If CFSE negative control is needed, remove cells now.		#If CFSE negative control is needed, remove cells now.
	#Make '2X' concentration (10 μM) in PBS		#Make '2X' concentration (10 μM) in PBS
	#*For example: add 5 mL PBS + 10 μL 5 mM CFSE		#*For example: add 5 mL PBS + 10 μL 5 mM CFSE
-	#Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube.	+	#Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
	#Place in 37°C H<sub>2</sub>O bath x 10 min.		#Place in 37°C H<sub>2</sub>O bath x 10 min.
	#Wash in 10 mL complete medium.		#Wash in 10 mL complete medium.
-	#Resuspend in medium at desired density	+	#Resuspend in medium at desired density

	==Discussion==		==Discussion==

Wayne G. Shreffler: CFSE Labeling moved to Shreffler:CFSE Labeling: lab protocol

Wayne G. Shreffler — Wed, 20 May 2009 03:45:34 GMT

CFSE Labeling moved to Shreffler:CFSE Labeling: lab protocol

←Older revision

Revision as of 03:45, 20 May 2009