Protocols/Template - Revision history

Sean P Corum at 21:27, 18 June 2012

2012-06-18T21:27:39Z

←Older revision		Revision as of 21:27, 18 June 2012
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	==Materials==		==Materials==
-	List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

-	*supply 1 (i.e. tubes of a ~~certain size? spreaders?)~~	+	For a 50 μL PCR reaction:
-	*reagent 1	+
-	*~~X μL reagent~~ 2	+	* 35 μL H<sub>2</sub>O
-	**~~component A~~ (~~reagent 2 is made up of multiple components~~)	+	* 5 μL 10X PCR buffer
-	**~~component B~~	+	* 5 μL 2mM dNTPs (each)
-	*~~equipment~~ 1	+	* 1.5 μL 50mM MgCl<sub>2</sub>
-	*~~equipment 2~~	+	* 1 μL 50μM sense primer
		+	* 1 μL 50μM antisense primer
		+	* 1 μL 5nM DNA template
		+	* 0.5 μL TAQ DNA polyermerase

	==Procedure==		==Procedure==
-	#~~Step 1~~	+	# In a PCR tube, mix the components on ice in the order they are listed above.
-	#~~Step 2~~	+	# Perform thermocycling program
-	#*Step 2 has some additional information that goes with it. i.e. Keep at 4°C.	+	## 95 °C 5 min
-	#~~Step 3~~	+	## 95 °C 30 s
-	##~~Step 3 has multiple~~ sub~~-steps within it.~~	+	## T<sub>H</sub> 30 s
-	##~~Enumerate~~ each of ~~those~~.	+	## 72 °C 1 min for each 1 kb PCR product
		+	## Repeat steps 2-4 a total of 12-36 times (24 is standard).
		+	## 72 °C 5 min
		+	## 12 °C hold

	==Notes==		==Notes==

Kathryn Muratore: Fixing broken biblio section

2011-08-15T21:57:09Z

Fixing broken biblio section

←Older revision		Revision as of 21:57, 15 August 2011
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	==References==		==References==
	'''Relevant papers and books'''		'''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, but this format doesn't work currently.
	<biblio>		<biblio>
	#Goldbeter-PNAS-1981 pmid=6947258		#Goldbeter-PNAS-1981 pmid=6947258
	#Jacob-JMB-1961 pmid=13718526		#Jacob-JMB-1961 pmid=13718526
	#Ptashne-Genetic-Switch isbn=0879697164		#Ptashne-Genetic-Switch isbn=0879697164
-	</biblio>	+	</biblio>-->
		+	<!-- Try the [[Template:FormatRef\|FormatRef template]]-->
		+	#{{FormatRef\|Goldbeter, A and Koshland, DE\|1981\| \|Proc Natl Acad Sci U S A 78(11)\|6840-4\| }} PMID 6947258
		+	#{{FormatRef\|Jacob, F and Monod, J J\|1961\| \|Mol Biol 3(3)\|318-56\| }} PMID 13718526
		+	#{{FormatRef\|Ptashne, M\|2004\|Genetic Switch: Phage Lambda Revisited\| \| \|Cold Spring Harbor Laboratory Press}} ISBN 0879697164

	==Contact==		==Contact==

Kathryn Muratore: reverting to clean template after significant changes made by User:Kehinde Kassim

2011-08-15T21:43:15Z

reverting to clean template after significant changes made by User:Kehinde Kassim

Show changes

Kehinde Kassim: /* This is a sterile procedure and all steps should be performed in a hood. /

2009-10-24T22:25:45Z

*This is a sterile procedure and all steps should be performed in a hood.

←Older revision		Revision as of 22:25, 24 October 2009
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	# After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes. Aspirate supernatant after centrifugation.		# After incubation, wash the CFSE stained cells in 10mL of AIM-V @ 300g for 10 minutes. Aspirate supernatant after centrifugation.
	# Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL. For plating, each well should contain 2 milliion cells/mL.		# Resuspend CFSE stained cells in AIM-V medium @ 4 million cells/mL. For plating, each well should contain 2 milliion cells/mL.
-	* Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube. For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2. Vortex gently.	+	* Begin the stimulation process by preparing AIM-V medium + a 2X solution of IL-2 by adding 2μL of IL-2 per mL of medium in a 15mL conical tube. For 5 stimulant conditions, you will need atleast 2.5mL of AIM-V + IL-2. Vortex gently.

	# Prepare solutions for each stimulant condition in 5 mL PP tubes as follows:		# Prepare solutions for each stimulant condition in 5 mL PP tubes as follows:

Kehinde Kassim: /* T REGULATORY CELL SURFACE STAINING */

2009-10-24T22:24:37Z

T REGULATORY CELL SURFACE STAINING

←Older revision		Revision as of 22:24, 24 October 2009
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	* At this point in the experiment, one can fix/freeze the cells or continue with Intracellular cytokine staining.		* At this point in the experiment, one can fix/freeze the cells or continue with Intracellular cytokine staining.

-	## To fix/freeze the cells, pipette 500μl of 1X Facs Lysis Buffer into each tube and incubate @ 25°C in the dark for 15 minutes. After the incubation, pipette 500μl of the 20% DMSO + PBS solution into each tube.	+	# To fix/freeze the cells, pipette 500μl of 1X Facs Lysis Buffer into each tube and incubate @ 25°C in the dark for 15 minutes. After the incubation, pipette 500μl of the 20% DMSO + PBS solution into each tube.

-	# Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C until when you are ready to conduct Intracellular cytokine staining..	+	# Transfer the 1ml of cells + 1X Facs Lysis Buffer + 20% DMSO/PBS solution into an appropriately labeled cluster tubes, and freeze at -80°C until when you are ready to conduct Intracellular cytokine staining..

	# To Intracellular Cytokine stain the cell with Fox P3, prepare a Fix/Perm working solution as follow: dilute the Fix/Perm Concentrate (1 part) into the Fix/Perm Diluent (3 parts) to the desired volume of working solution (1mL per tube).		# To Intracellular Cytokine stain the cell with Fox P3, prepare a Fix/Perm working solution as follow: dilute the Fix/Perm Concentrate (1 part) into the Fix/Perm Diluent (3 parts) to the desired volume of working solution (1mL per tube).

-	# Resuspend cell pellet with pulse vortex and add 1mL of Fix/Perm buffer to each sample tube.	+	# Resuspend cell pellet with pulse vortex and add 1mL of Fix/Perm buffer to each sample tube. Pulse vortex again.
-	Pulse vortex again.	+

-	# Incubate at 4°C for 30-60 minutes in the dark (preferably for 60 minutes if possible).	+	# Incubate at 4°C for 30-60 minutes in the dark (preferably for 60 minutes if possible). Wash for 10 minutes at 800 g x 2 (4°C) with 2mL of 1X Perm Buffer (made from 10X solution using dH2O). Decant supernatant.
-	Wash for 10 minutes at 800 g x 2 (4°C) with 2mL of 1X Perm Buffer (made from 10X solution using dH2O). Decant supernatant.	+

-	# Add 20μL of Foxp3 antibody into 100μL of 1X Perm Buffer. Incubate for at least 30 minutes (45	+	# Add 20μL of Foxp3 antibody into 100μL of 1X Perm Buffer. Incubate for at least 30 minutes (45 minutes if possible) at 4°C.
-	minutes if possible) at 4°C.	+

-	# Wash at ~~800 g~~ for 10 minutes x 2 with 2mL 1X Perm Buffer. Decant supernatant. Vortex and acquire using the Flow machine at 17-40.	+	# Wash at 800g for 10 minutes x 2 with 2mL 1X Perm Buffer. Decant supernatant. Vortex and acquire using the Flow machine at 17-40.

	==Notes==		==Notes==

Kehinde Kassim: /* COLLECTION OF SUPERNATANTS */

2009-10-24T22:21:28Z

COLLECTION OF SUPERNATANTS

←Older revision		Revision as of 22:21, 24 October 2009
Line 190:		Line 190:
	### Stimulant 4 = Tetanus Toxoid		### Stimulant 4 = Tetanus Toxoid

		+	# For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the PS culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.

-	## For each stimulant, using a 1000μL pipette tip, transfer 800μL of supernatant from the PS culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.	+	# Cap the cluster tubes and store in the –80oC freezer (by the freight elevators).
-		+
-	## Cap the cluster tubes and store in the –80oC freezer (by the freight elevators).	+

	=='''T REGULATORY CELL SURFACE STAINING'''==		=='''T REGULATORY CELL SURFACE STAINING'''==

Kehinde Kassim: /* Overview */

2009-10-24T22:19:58Z

Overview

←Older revision		Revision as of 22:19, 24 October 2009
Line 17:		Line 17:
	==Overview==		==Overview==

-	* This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.	+	This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.

	==Materials==		==Materials==

Kehinde Kassim: /* Overview */

2009-10-24T22:19:16Z

Overview

←Older revision		Revision as of 22:19, 24 October 2009
Line 17:		Line 17:
	==Overview==		==Overview==

-	This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.	+	* This is a protocol designed for T-regulatory assay for Mouse allergen (Mus m1) study.

	==Materials==		==Materials==

Kehinde Kassim: /* This is a sterile procedure and all steps should be performed in a hood. /

2009-10-24T22:16:24Z

*This is a sterile procedure and all steps should be performed in a hood.

←Older revision		Revision as of 22:16, 24 October 2009
Line 132:		Line 132:
	# After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10^6/ml in 15 ml PP conical tube.		# After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10^6/ml in 15 ml PP conical tube.

		+	=='''PBMC Antigen Stimulation Assay'''==
	==*This is a sterile procedure and all steps should be performed in a hood.==		==*This is a sterile procedure and all steps should be performed in a hood.==
	# Remove stimulants from the freezer and thaw.		# Remove stimulants from the freezer and thaw.

Kehinde Kassim: /* PBMC Antigen Stimulation Assay */

2009-10-24T22:15:24Z

PBMC Antigen Stimulation Assay

←Older revision		Revision as of 22:15, 24 October 2009
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	=='''Procedure'''==		=='''Procedure'''==

-	~~=='''PBMC Antigen Stimulation Assay'''==~~	+
		+
	==Isolation of Mononuclear Cells==		==Isolation of Mononuclear Cells==

Protocols/Template - Revision history

Sean P Corum at 21:27, 18 June 2012

Kathryn Muratore: Fixing broken biblio section

Kathryn Muratore: reverting to clean template after significant changes made by User:Kehinde Kassim

Kehinde Kassim: /* *This is a sterile procedure and all steps should be performed in a hood. */

Kehinde Kassim: /* T REGULATORY CELL SURFACE STAINING */

Kehinde Kassim: /* COLLECTION OF SUPERNATANTS */

Kehinde Kassim: /* Overview */

Kehinde Kassim: /* Overview */

Kehinde Kassim: /* *This is a sterile procedure and all steps should be performed in a hood. */

Kehinde Kassim: /* PBMC Antigen Stimulation Assay */

Kehinde Kassim: /* This is a sterile procedure and all steps should be performed in a hood. /

Kehinde Kassim: /* This is a sterile procedure and all steps should be performed in a hood. /