Shreffler:JAX Tcell studies - Revision history

Wayne G. Shreffler: /* Collection of Cell Pellet for RNA Isolation */

Wayne G. Shreffler — Wed, 26 Jan 2011 18:03:17 GMT

Collection of Cell Pellet for RNA Isolation

←Older revision		Revision as of 18:03, 26 January 2011
Line 109:		Line 109:
	#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil		#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil
	# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.		# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.
-	# Centrifuge cells at 400 g for 5 minutes at ~~4°C~~. Please take special care to minimize disturbing the cell pellet.	+	# Centrifuge cells at 400 g for 5 minutes at RT. Please take special care to minimize disturbing the cell pellet.
	# Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.		# Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.
	# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.		# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.

Wayne G. Shreffler: /* Collection of Supernatants */

Wayne G. Shreffler — Wed, 26 Jan 2011 18:02:51 GMT

Collection of Supernatants

←Older revision		Revision as of 18:02, 26 January 2011
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	# Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.		# Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.
	# Note variations in cell density, obvious contamination.		# Note variations in cell density, obvious contamination.
-	# Centrifuge tubes at ~~300g~~ for 5 minutes at 25°C.	+	# Centrifuge tubes at 400g for 5 minutes at 25°C.
	# Obtain a cluster tube rack for the storage of supernatants.		# Obtain a cluster tube rack for the storage of supernatants.
	#: Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.		#: Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.

Wayne G. Shreffler: /* PBMC Stimulation */

Wayne G. Shreffler — Wed, 23 Jun 2010 15:05:11 GMT

PBMC Stimulation

←Older revision		Revision as of 15:05, 23 June 2010
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	## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.		## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
	# Prepare solutions for each stimulant condition as follows:		# Prepare solutions for each stimulant condition as follows:
-	#* ~~200~~ μg/mL MusM1 in AIM-V:	+	#* 10 μg/mL MusM1 in AIM-V:
	#** place 450 μL of AIM-V in the appropriately labeled tube		#** place 450 μL of AIM-V in the appropriately labeled tube
	#** add 50 μL of allergen (MusM1)		#** add 50 μL of allergen (MusM1)
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	#** add 500 μL of cell suspension		#** add 500 μL of cell suspension
	#** pipette up and down to mix		#** pipette up and down to mix
-	#* ~~1 μg/mL~~ anti-CD3, anti-CD28 beads in AIM-V:	+	#* anti-CD3, anti-CD28 beads in AIM-V:
	#** place 500 μL of AIM-V in the appropriately labeled tube		#** place 500 μL of AIM-V in the appropriately labeled tube
	#** add 2.5 μL of CD3, CD28 expander beads		#** add 2.5 μL of CD3, CD28 expander beads

Cristy Benson: /* PBMC Stimulation */

Cristy Benson — Mon, 17 May 2010 19:29:32 GMT

PBMC Stimulation

←Older revision		Revision as of 19:29, 17 May 2010
Line 56:		Line 56:
	==PBMC Stimulation==		==PBMC Stimulation==
	'''(Sterile Technique)'''		'''(Sterile Technique)'''
-	# Remove ~~stimulants~~ from the freezer and thaw.	+	# Remove Musm1 from the freezer and thaw.
		+	# Centrifuge the Musm1 briefly in the small centrifuge.
	# Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).		# Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).
	## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.		## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.

Cristy Benson: /* Materials */

Cristy Benson — Mon, 17 May 2010 19:23:41 GMT

Materials

Wayne G. Shreffler: /* Collection of Cell Pellet for RNA Isolation */

Wayne G. Shreffler — Wed, 07 Apr 2010 16:33:15 GMT

Collection of Cell Pellet for RNA Isolation

←Older revision		Revision as of 16:33, 7 April 2010
Line 113:		Line 113:
	'''(non-sterile technique)'''		'''(non-sterile technique)'''

-	# Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL ~~of ~14.3M stock solution~~ of β-mercaptoethanol per 1ml of the RLT buffer.	+	# Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of β-mercaptoethanol stock solution per 1ml of the RLT buffer.
	#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil		#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil
	# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.		# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.

Wayne G. Shreffler: /* PBMC Stimulation */

Wayne G. Shreffler — Wed, 07 Apr 2010 16:28:10 GMT

PBMC Stimulation

←Older revision		Revision as of 16:28, 7 April 2010
Line 70:		Line 70:
	## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.		## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.
	# Prepare solutions for each stimulant condition as follows:		# Prepare solutions for each stimulant condition as follows:
-	#* 200 μg/mL MusM1 in AIM-V: place 450 μL of AIM-V in the appropriately labeled tube ~~and~~ add 50 μL of allergen (MusM1)~~. Then~~ add 500 μL of cell suspension~~. Pipette~~ up and down.	+	#* 200 μg/mL MusM1 in AIM-V:
-	#* AIM-V medium alone: place 500 μL of AIM-V in the appropriately labeled tube ~~and~~ add 500 μL of cell suspension~~. Pipette~~ up and down.	+	#** place 450 μL of AIM-V in the appropriately labeled tube
-	#* 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V: place 500 μL of AIM-V in the appropriately labeled tube ~~and~~ add 2.5 μL of CD3, CD28 expander beads~~. Then~~ add 500 μL of cell suspension~~. Pipette~~ up and down.	+	#** add 50 μL of allergen (MusM1)
-	#* Place the culture tubes in the 37°C CO<sub>2</sub> incubator for 48 hours.	+	#** add 500 μL of cell suspension
		+	#** pipette up and down to mix
		+	#* AIM-V medium alone:
		+	#** place 500 μL of AIM-V in the appropriately labeled tube
		+	#** add 500 μL of cell suspension
		+	#** pipette up and down to mix
		+	#* 1 μg/mL anti-CD3, anti-CD28 beads in AIM-V:
		+	#** place 500 μL of AIM-V in the appropriately labeled tube
		+	#** add 2.5 μL of CD3, CD28 expander beads
		+	#** add 500 μL of cell suspension
		+	#** pipette up and down to mix
		+	#* Place the culture tubes '''with tube caps loose''' in the 37°C CO<sub>2</sub> incubator for 48 hours.

	==Collection of Supernatants==		==Collection of Supernatants==

Wayne G. Shreffler: /* PBMC Stimulation */

Wayne G. Shreffler — Wed, 07 Apr 2010 16:19:09 GMT

PBMC Stimulation

←Older revision		Revision as of 16:19, 7 April 2010
Line 65:		Line 65:
	'''(Sterile Technique)'''		'''(Sterile Technique)'''
	# Remove stimulants from the freezer and thaw.		# Remove stimulants from the freezer and thaw.
-	# Label 5 mL sterile polypropylene tubes~~, each well~~ with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).	+	# Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).
	## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.		## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.
	## AIM-V (negative control): AIM-V medium alone.		## AIM-V (negative control): AIM-V medium alone.

Wayne G. Shreffler: /* Collection of Cell Pellet for RNA Isolation */

Wayne G. Shreffler — Wed, 07 Apr 2010 15:50:02 GMT

Collection of Cell Pellet for RNA Isolation

←Older revision		Revision as of 15:50, 7 April 2010
Line 109:		Line 109:
	# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.		# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.
	# Vortex each tube twice for 15 seconds each time.		# Vortex each tube twice for 15 seconds each time.
-	~~# Centrifuge briefly at 1,000g for few seconds to collect all liquid at the bottom of the tube.~~
	# Transfer to labeled cryovial tube.		# Transfer to labeled cryovial tube.
	# Freeze immediately and store for shipment.		# Freeze immediately and store for shipment.

Wayne G. Shreffler: /* Collection of Cell Pellet for RNA Isolation */

Wayne G. Shreffler — Wed, 07 Apr 2010 15:49:45 GMT

Collection of Cell Pellet for RNA Isolation

←Older revision		Revision as of 15:49, 7 April 2010
Line 102:		Line 102:
	'''(non-sterile technique)'''		'''(non-sterile technique)'''

-	~~# Add 1mL of staining buffer to the cell pellet of each culture tube, vortex on low.~~	+	# Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of ~14.3M stock solution of β-mercaptoethanol per 1ml of the RLT buffer.
-	# Centrifuge cells at 400 g for 5 minutes at 4°C. Please take special care to minimize the cell pellet disturbing or lost. Use a 1000 μL pipetman to aspirate most of the supernatant removing the last ~100 μL with a 200 μL pipetman. Make sure that only filtered pre-sterilized tips are being used from this step and later.	+
-	# Prepare RLT buffer containing β-mercaptoethanol by adding 10 μL of ~14.3M stock solution of β-mercaptoethanol per 1ml of the RLT buffer	+
	#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil		#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil
		+	# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.
		+	# Centrifuge cells at 400 g for 5 minutes at 4°C. Please take special care to minimize disturbing the cell pellet.
		+	# Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.
	# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.		# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.
	# Vortex each tube twice for 15 seconds each time.		# Vortex each tube twice for 15 seconds each time.

←Older revision		Revision as of 19:23, 17 May 2010
Line 7:		Line 7:
	*On average, 18mL of whole heparinized blood/patient will be given.		*On average, 18mL of whole heparinized blood/patient will be given.
	*target to isolate 8 x 10<sup>6</sup> (8 million) cells for 48 hour culture		*target to isolate 8 x 10<sup>6</sup> (8 million) cells for 48 hour culture
-	*Phosphate buffered saline (PBS)
-
	*Ficoll Paque Plus [Endotoxin tested], room temperature.		*Ficoll Paque Plus [Endotoxin tested], room temperature.
	*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)		*Staining buffer (PBS + 5g BSA/L + 2mM EDTA)
-
	*Sterile phosphate buffered saline (PBS), room temperature		*Sterile phosphate buffered saline (PBS), room temperature
	*AIM V Media (w/HSA). Invitrogen # 0870112DK (Expires 1 month after opening)		*AIM V Media (w/HSA). Invitrogen # 0870112DK (Expires 1 month after opening)
-	*Cluster tubes or Cryovial tubes ~~and racks.~~	+	*Cluster tubes
-		+	*Cryovial tubes
	*Turk Blood Diluting Fluid (RICCA # 8850-16).		*Turk Blood Diluting Fluid (RICCA # 8850-16).
	*Sterile conical tubes (15mL, 50mL)		*Sterile conical tubes (15mL, 50mL)
	*CD3, CD28 expander beads (Invitrogen 111-61D)		*CD3, CD28 expander beads (Invitrogen 111-61D)
-
	*Sterile, graduated transfer pipettes.		*Sterile, graduated transfer pipettes.
	*Mus M1 (50μL) aliquot tube.		*Mus M1 (50μL) aliquot tube.
-
-	*A solution of 20% DMSO + PBS.
-
	*Hemocytometer or disposable slides for the automated counter or manual counter		*Hemocytometer or disposable slides for the automated counter or manual counter
-
	*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)		*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)
	*Sterile 5mL polypropylene round-bottom tubes		*Sterile 5mL polypropylene round-bottom tubes