Shreffler:JAX Basophil Activation - Revision history

Alex Ma: /* Procedure */

2011-01-11T18:27:49Z

Procedure

←Older revision		Revision as of 18:27, 11 January 2011
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	# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).		# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).
	# Prepare Basophil medium (BM) as follows:		# Prepare Basophil medium (BM) as follows:
-	#* Add 5 μL of IL-3 into 2.5 mL of warm BM (use 5 mL PP tube). '''Vortex'''	+	#* Add 5 μL of IL-3 into 2.5 mL of warm RPMI (use 5 mL PP tube). '''Vortex'''
	# Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP.		# Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP.
	# Add 135 μL of warm BM to each of the Calcium Ionophore and PMA tubes.		# Add 135 μL of warm BM to each of the Calcium Ionophore and PMA tubes.
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	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
	# Ship to Shreffler Lab at room temperature.		# Ship to Shreffler Lab at room temperature.
		+	**Note that IL-3 stock at JAX is different concentration than at Shrefflab

	==Discussion==		==Discussion==

Wayne G. Shreffler: /* Procedure */

2010-12-15T16:57:33Z

Procedure

←Older revision		Revision as of 16:57, 15 December 2010
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	# Place RPMI in a 37°C water bath for a minimum of 10 minutes.		# Place RPMI in a 37°C water bath for a minimum of 10 minutes.
	# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).		# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).
-	# Prepare Basophil medium ~~as follows~~ (~~set aside for step 10~~):	+	# Prepare Basophil medium (BM) as follows:
-	#* Add 4 μL of IL-3 into 2.0 mL of warm ~~RPMI~~ (use 5 mL PP tube). '''Vortex'''	+	#* Add 5 μL of IL-3 into 2.5 mL of warm BM (use 5 mL PP tube). '''Vortex'''
-	# Add 270 μL of warm ~~RPMI~~ to the following stimulants: anti-IgE and fMLP.	+	# Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP.
-	# Add 135 μL of warm ~~RPMI~~ to each of the Calcium Ionophore and PMA tubes.	+	# Add 135 μL of warm BM to each of the Calcium Ionophore and PMA tubes.
	# Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA. '''Vortex'''		# Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA. '''Vortex'''
	# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
-	#* Add 270 μL ~~of prepared Basophil medium~~ to Mouse 1 stimulant. '''Vortex'''	+	#* Add 270 μL BM to Mouse 1 stimulant. '''Vortex'''
-	#* ~~Transfer~~ 270 μL ~~of the Basophil medium prepared above~~ to each of the eppendorf tubes labeled Mouse 2-5.	+	#* Add 270 μL BM to each of the eppendorf tubes labeled Mouse 2-5.
	#* Transfer 30 μL from Mouse 1 stimulant into Mouse 2 eppendorf, '''vortex'''. Repeat this from Mouse 2 eppendorf into Mouse 3 eppendorf		#* Transfer 30 μL from Mouse 1 stimulant into Mouse 2 eppendorf, '''vortex'''. Repeat this from Mouse 2 eppendorf into Mouse 3 eppendorf
	#* Continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 –Mouse 5). '''Vortex''' after each dilution.		#* Continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 –Mouse 5). '''Vortex''' after each dilution.

Alex Ma: /* Procedure */

2010-10-26T17:24:57Z

Procedure

←Older revision		Revision as of 17:24, 26 October 2010
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	# Centrifuge for 5 minutes at 800g, RT.		# Centrifuge for 5 minutes at 800g, RT.
	# Aspirate supernatant, taking care not to disturb pellet (leaving some residual supernatant is okay)		# Aspirate supernatant, taking care not to disturb pellet (leaving some residual supernatant is okay)
-	# Resuspend pellet in 50 μL staining buffer, making total liquid volume approximately ~~200~~ μL.	+	# Resuspend pellet in 50 μL staining buffer, making total liquid volume approximately 300 μL.
	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
	# Ship to Shreffler Lab at room temperature.		# Ship to Shreffler Lab at room temperature.

Alex Ma: /* Procedure */

2010-10-04T17:41:07Z

Procedure

←Older revision		Revision as of 17:41, 4 October 2010
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	# Top off tube with staining buffer		# Top off tube with staining buffer
	# Centrifuge for 5 minutes at 800g, RT.		# Centrifuge for 5 minutes at 800g, RT.
-	# Aspirate supernatant, taking care not	+	# Aspirate supernatant, taking care not to disturb pellet (leaving some residual supernatant is okay)
-	# Resuspend pellet in 50 μL staining buffer.	+	# Resuspend pellet in 50 μL staining buffer, making total liquid volume approximately 200 μL.
	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
	# Ship to Shreffler Lab at room temperature.		# Ship to Shreffler Lab at room temperature.

Alex Ma: /* Procedure */

2010-10-04T17:39:24Z

Procedure

←Older revision		Revision as of 17:39, 4 October 2010
Line 69:		Line 69:
	# Top off tube with staining buffer		# Top off tube with staining buffer
	# Centrifuge for 5 minutes at 800g, RT.		# Centrifuge for 5 minutes at 800g, RT.
-	# ~~Decant~~ supernatant ~~(do~~ not ~~double dump).~~	+	# Aspirate supernatant, taking care not
	# Resuspend pellet in 50 μL staining buffer.		# Resuspend pellet in 50 μL staining buffer.
	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.

Alex Ma: /* Procedure */

2010-09-13T09:41:13Z

Procedure

←Older revision		Revision as of 09:41, 13 September 2010
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	# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''		# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''
	# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:		# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
-	#* To 800 uL staining buffer, add 360 μL of premade antibody ~~cocktail~~ sent by Shreffler Lab containing:	+	#* To 800 uL staining buffer, add 360 μL of premade antibody mix sent by Shreffler Lab containing:
	#* CD63 - FITC (9 uL per test)		#* CD63 - FITC (9 uL per test)
	#* CD203c - PE-Cy7 (9 uL per test)		#* CD203c - PE-Cy7 (9 uL per test)

Alex Ma: /* Procedure */

2010-09-13T09:39:45Z

Procedure

←Older revision		Revision as of 09:39, 13 September 2010
Line 49:		Line 49:
	# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''		# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''
	# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:		# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until you are ready to use it:
-	#* To ~~900~~ uL staining buffer, add 90 μL ~~each~~ of:	+	#* To 800 uL staining buffer, add 360 μL of premade antibody cocktail sent by Shreffler Lab containing:
-	#* CD63	+	#* CD63 - FITC (9 uL per test)
-	#* CD203c	+	#* CD203c - PE-Cy7 (9 uL per test)
-	#* CD123	+	#* CD123 - PE-Cy5 (9 uL per test)
-	~~#: and 12 μL of:~~	+	#* HLA-DR - APC (4.5 uL per test)
-	#* HLA-DR	+	#* CRTH2 - PE (4.5 uL per test)
	#: '''Vortex'''		#: '''Vortex'''
	# After the 20 minutes of incubation, remove the samples.		# After the 20 minutes of incubation, remove the samples.

Cristy Benson: /* Procedure */

2010-05-17T19:32:01Z

Procedure

←Older revision		Revision as of 19:32, 17 May 2010
Line 28:		Line 28:
	# Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer.		# Remove pre-made stimulants (fMLP, Mouse allergen, CaI, PMA, Anti-IgE) from freezer.
	# Label each stimulant and put aside to thaw.		# Label each stimulant and put aside to thaw.
		+	# Centrifuge stimulants in the small centrifuge briefly.
	# Aliquot 5 mL of RPMI into 15 mL conical tube		# Aliquot 5 mL of RPMI into 15 mL conical tube
	# Place RPMI in a 37°C water bath for a minimum of 10 minutes.		# Place RPMI in a 37°C water bath for a minimum of 10 minutes.

Wayne G. Shreffler: /* Procedure */

2010-04-14T19:10:08Z

Procedure

←Older revision		Revision as of 19:10, 14 April 2010
Line 69:		Line 69:
	# Centrifuge for 5 minutes at 800g, RT.		# Centrifuge for 5 minutes at 800g, RT.
	# Decant supernatant (do not double dump).		# Decant supernatant (do not double dump).
-	# Resuspend pellet in ~~100~~ μL staining buffer.	+	# Resuspend pellet in 50 μL staining buffer.
	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
	# Ship to Shreffler Lab at room temperature.		# Ship to Shreffler Lab at room temperature.

Wayne G. Shreffler: /* Procedure */

2010-04-06T18:04:05Z

Procedure

←Older revision		Revision as of 18:04, 6 April 2010
Line 32:		Line 32:
	# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).		# Label five eppendorf tubes for intermediate CaI/PMA preparation and Mouse 2-5, and eleven 5 mL PP tubes for conditions (A-K).
	# Prepare Basophil medium as follows (set aside for step 10):		# Prepare Basophil medium as follows (set aside for step 10):
-	#* Add 4 μL of IL-3 into 2.0 mL of warm RPMI (use 5 mL PP tube).	+	#* Add 4 μL of IL-3 into 2.0 mL of warm RPMI (use 5 mL PP tube). '''Vortex'''
-	#* '''Vortex'''	+
	# Add 270 μL of warm RPMI to the following stimulants: anti-IgE and fMLP.		# Add 270 μL of warm RPMI to the following stimulants: anti-IgE and fMLP.
	# Add 135 μL of warm RPMI to each of the Calcium Ionophore and PMA tubes.		# Add 135 μL of warm RPMI to each of the Calcium Ionophore and PMA tubes.
-	# Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA.	+	# Transfer both diluted stimulants into a single intermediate 1.5 mL tube labeled CaI/PMA. '''Vortex'''
-	# '''Vortex'''	+
	# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Mouse 1 stimulant as follows:
-	#* Add 270 μL of prepared Basophil medium to Mouse 1 stimulant.	+	#* Add 270 μL of prepared Basophil medium to Mouse 1 stimulant. '''Vortex'''
-	#* Transfer 270 μL of the Basophil medium prepared above to each of the Mouse 2-5 ~~eppendorf tubes~~.	+	#* Transfer 270 μL of the Basophil medium prepared above to each of the eppendorf tubes labeled Mouse 2-5.
-	#* Transfer 30 μL from Mouse 1 stimulant into Mouse 2 eppendorf, '''vortex'''. Repeat this from Mouse 2 eppendorf into Mouse 3 eppendorf ~~and continue~~ making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 –Mouse 5). '''Vortex''' after each dilution.	+	#* Transfer 30 μL from Mouse 1 stimulant into Mouse 2 eppendorf, '''vortex'''. Repeat this from Mouse 2 eppendorf into Mouse 3 eppendorf
		+	#* Continue making 10-fold dilutions in the same manner until all four Mouse dilutions have been prepared (Mouse 2 –Mouse 5). '''Vortex''' after each dilution.
	# Transfer 250 μL of warm RPMI into tubes A and B.		# Transfer 250 μL of warm RPMI into tubes A and B.
-	# Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (mouse allergen for tube 'G'; basophil medium for tube 'C').	+	# Transfer 250 μL from each of the prepared stimulants above, to the corresponding PP tubes (mouse allergen for tube 'G'; basophil medium for tube 'C', etc.).
	#;[[Image:jax_stim.tiff]]		#;[[Image:jax_stim.tiff]]
		+	# Gently mix blood by inverting green top tube 4 times.
	# '''IN THE HOOD''', add 250 μL of patient blood to all eleven tubes.		# '''IN THE HOOD''', add 250 μL of patient blood to all eleven tubes.
	# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''		# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>). '''Do not shake tubes!'''
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	#* HLA-DR		#* HLA-DR
	#: '''Vortex'''		#: '''Vortex'''
-	# After 20 minutes of incubation, remove the samples ~~and~~ add 50 μL cold PBS ~~w/ 20 mM~~ EDTA to each tube.	+	# After the 20 minutes of incubation, remove the samples.
		+	# Immediately add 50 μL cold PBS-EDTA to each tube.
	# Add 110 μL of antibody cocktail to tubes B -> K '''(DO NOT ADD TO A!)'''.		# Add 110 μL of antibody cocktail to tubes B -> K '''(DO NOT ADD TO A!)'''.
		+	# Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
	# Incubate for 30 min at 4°C. '''Protect from light'''.		# Incubate for 30 min at 4°C. '''Protect from light'''.
-	# ~~Wash samples with~~ 2 mL staining buffer ~~and centrifuge~~ for 5 minutes at 300g, RT	+	# Add 2 mL staining buffer.
		+	# Invert tubes 3X to mix
		+	# Centrifuge for 5 minutes at 300g, RT
	# Aspirate supernatant, taking care not to disturb cell pellet.		# Aspirate supernatant, taking care not to disturb cell pellet.
-	# Add 2 mL of 1x FACS lysing solution~~. Cover with parafilm~~ and '''invert repeatedly until pellet is fully resuspended'''. ~~(An alternative is to add the FACS while tube is on lighter speed vortex. Watch to make sure mixing is thorough; by the time FACS is added to last tube, first tube should be noticeably less opaque.)~~	+	# Add 3 mL of 1x FACS lysing solution one tube at a time, recap and '''invert repeatedly until pellet is fully resuspended'''.
	# Place tubes in the dark for 15 minutes or overnight in the refrigerator.		# Place tubes in the dark for 15 minutes or overnight in the refrigerator.
-	# ~~Wash samples~~ with ~~1.5 mL~~ staining buffer~~, then centrifuge~~ for 5 minutes at 800g, RT. Decant supernatant ~~and resuspend~~ pellet in 100 μL staining buffer.	+	# Top off tube with staining buffer
		+	# Centrifuge for 5 minutes at 800g, RT.
		+	# Decant supernatant (do not double dump).
		+	# Resuspend pellet in 100 μL staining buffer.
	# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.		# Transfer to 1.5 mL eppendorf tube and seal with parafilm for shipping.
	# Ship to Shreffler Lab at room temperature.		# Ship to Shreffler Lab at room temperature.