Isolation of murine splenocytes - Revision history

Torsten Waldminghaus at 17:20, 23 February 2009

2009-02-23T17:20:40Z

←Older revision		Revision as of 17:20, 23 February 2009
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		+	{{back to protocols}}
	==Overview==		==Overview==

Reshma P. Shetty at 18:39, 9 May 2007

2007-05-09T18:39:26Z

←Older revision		Revision as of 18:39, 9 May 2007
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	#CP3 Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)		#CP3 Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)
	</biblio>		</biblio>
		+
		+	[[Category:Protocol]]
		+	[[Category:Mouse]]

Kurt: /* Protocol B */

2006-10-19T16:28:23Z

Protocol B

←Older revision		Revision as of 16:28, 19 October 2006
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	#Homogenize the spleen between the frosted ends of the slides.		#Homogenize the spleen between the frosted ends of the slides.
	#Pass the homogenized spleen through the cell strainer (or nylon mesh) mounted on a 50mL conical.		#Pass the homogenized spleen through the cell strainer (or nylon mesh) mounted on a 50mL conical.
-	#Continue with step 4 of Procedure B.	+	#Continue with step 4 of Procedure A.

	==Notes==		==Notes==

Kurt: /* Reagents */

2006-09-26T06:01:06Z

Reagents

←Older revision		Revision as of 06:01, 26 September 2006
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	**1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM)		**1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM)
	**100mL fetal bovine/calf serum		**100mL fetal bovine/calf serum
-	**10mL ~~100x~~ PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016)	+	**10mL 100X PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016)
	***sterilize using 0.2μm filter; store at 4°		***sterilize using 0.2μm filter; store at 4°
	*ACK lysis buffer (1mL)		*ACK lysis buffer (1mL)
-	**1L deionized ~~H2O~~	+	**1L deionized water
	**8.29g NH4Cl		**8.29g NH4Cl
	**1g KHCO3		**1g KHCO3
-	**37.2mg ~~Na2EDTA~~	+	**37.2mg Na2-EDTA
	***pH solution to 7.2-7.4; sterilize using 0.2μm filter; store at 4°		***pH solution to 7.2-7.4; sterilize using 0.2μm filter; store at 4°
	*70% ethanol		*70% ethanol

Kurt at 04:41, 26 September 2006

2006-09-26T04:41:41Z

←Older revision		Revision as of 04:41, 26 September 2006
Line 1:		Line 1:
	==Overview==		==Overview==

-	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, Unit 1.9 ~~[[#References]]~~).	+	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, Unit 1.9 <cite>CP1</cite>).

	==Materials==		==Materials==
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	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->		<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
	<biblio>		<biblio>
-	#~~online~~ Current Protocols in Immunology, Unit 1.9: Removal of Lymphoid Organs [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0109/frame.html# link] (subscription required)	+	#CP1 Current Protocols in Immunology, Unit 1.9: Removal of Lymphoid Organs [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0109/frame.html# link] (subscription required)
-	#~~online~~ Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)	+	#CP3 Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)
	</biblio>		</biblio>

Kurt: /* References */

2006-09-26T04:33:27Z

References

←Older revision		Revision as of 04:33, 26 September 2006
Line 83:		Line 83:
	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->		<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
	<biblio>		<biblio>
-	#online Current Protocols in Immunology, ~~<div id="~~Unit 1.9~~">Unit 1.9</div>~~: Removal of Lymphoid Organs [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0109/frame.html# link] (subscription required)	+	#online Current Protocols in Immunology, Unit 1.9: Removal of Lymphoid Organs [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0109/frame.html# link] (subscription required)
	#online Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)		#online Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)
	</biblio>		</biblio>

Kurt: /* References */

2006-09-26T04:32:46Z

References

←Older revision		Revision as of 04:32, 26 September 2006
Line 1:		Line 1:
	==Overview==		==Overview==

-	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, ~~[[#~~Unit 1.9]]).	+	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, Unit 1.9 [[#References]]).

	==Materials==		==Materials==

Kurt: /* References */

2006-09-26T04:28:47Z

References

←Older revision		Revision as of 04:28, 26 September 2006
Line 81:		Line 81:

	==References==		==References==
-	~~'''Relevant papers and books'''~~
	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->		<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
	<biblio>		<biblio>
		+	#online Current Protocols in Immunology, <div id="Unit 1.9">Unit 1.9</div>: Removal of Lymphoid Organs [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0109/frame.html# link] (subscription required)
	#online Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)		#online Current Protocols in Immunology, Unit 3.1: Isolation of Mouse Mononuclear Cells [http://www.mrw.interscience.wiley.com/cp/cpim/articles/im0301/frame.html link] (subscription required)
	</biblio>		</biblio>

Kurt: /* Overview */

2006-09-26T04:26:44Z

Overview

←Older revision		Revision as of 04:26, 26 September 2006
Line 1:		Line 1:
	==Overview==		==Overview==

-	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies.	+	In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells), it helps to make single-cell suspensions so that the cells can be manipulated ex vivo easily. This protocol suggests ways in which you can do this without a lot of equipment or expensive supplies. This protocol can also be used to make cell suspensions from other lymphoid organs, such as the thymus or lymph nodes (see Current Protocols in Immunology, [[#Unit 1.9]]).

	==Materials==		==Materials==

Kurt: /* Protocol A */

2006-09-26T04:03:47Z

Protocol A

←Older revision		Revision as of 04:03, 26 September 2006
Line 61:		Line 61:
	#Spin cells at 800xg for 3 minutes.		#Spin cells at 800xg for 3 minutes.
	#Discard supernatant and resuspend pellet in 1mL ACK lysis buffer. Incubate at RT for 5-10 minutes. Add 9mL DMEM-10 and spin as before.		#Discard supernatant and resuspend pellet in 1mL ACK lysis buffer. Incubate at RT for 5-10 minutes. Add 9mL DMEM-10 and spin as before.
-	#Discard supernatant and resuspend pellet in 3mL DMEM-10.	+	#Discard supernatant and resuspend pellet in 3mL DMEM-10, discarding any dead cell mass.
	#Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).		#Count cells (dilute 10μL cell suspension in trypan blue, and count with hemcytometer).