Shreffler:Cisplatin Allergen - Revision history

Alex Ma: /* Procedure */

2011-12-19T16:43:44Z

Procedure

←Older revision		Revision as of 16:43, 19 December 2011
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	# Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).		# Obtain whole blood specimens collected in sodium heparin tube (green top). Keep at RT until use (within 16 hours).
	# Remove pre-made stimulants (fMLP, Drug, Anti-IgE) from freezer and let thaw.		# Remove pre-made stimulants (fMLP, Drug, Anti-IgE) from freezer and let thaw.
-	# Place ~~5 mL~~ RPMI in water bath for ~5-10 minutes.	+	# Place RPMI BSA aliquot in water bath for ~5-10 minutes.
-	# Prepare 15 5 mL PP tubes for conditions (A-O):	+	# Prepare 20 5 mL PS tubes for conditions (A-T):
	#*A: Unstained		#*A: Unstained
	#*B: RPMI		#*B: RPMI
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	#*N: Cisplatin 4 (0.001X)		#*N: Cisplatin 4 (0.001X)
	#*O: Cisplatin 5 (0.0001X)		#*O: Cisplatin 5 (0.0001X)
		+	#*P: Oxyplatin 1 (1X)
		+	#*Q: Oxyplatin 2 (0.1X)
		+	#*R: Oxyplatin 3 (0.01X)
		+	#*S: Oxyplatin 4 (0.001X)
		+	#*T: Oxyplatin 5 (0.0001X)
	#Prepare RPMI w/IL-3 as follows:		#Prepare RPMI w/IL-3 as follows:
-	#*Add 4 μL of aliquot IL-3 (10 µL/mL) into ~~2.0~~ mL of warm RPMI ~~in 5 mL PP tube~~. Mix well by inverting or vortex	+	#*Add 8 μL of aliquot IL-3 (10 µL/mL) into 20 mL of warm RPMI. Mix well by inverting or vortex
	# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3		# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
	#* For "A: Unstained" and "B: RPMI," use RPMI (no IL-3)		#* For "A: Unstained" and "B: RPMI," use RPMI (no IL-3)
	#* For "C: IL-3," use RPMI IL-3 as is.		#* For "C: IL-3," use RPMI IL-3 as is.
-	#* For "D: fMLP," add 30 µL of ~~4 mM~~ stock to ~~2370~~ µL RPMI IL-3 ~~(1 part in 80), then add 20 µL of this dilution with 980 µL RPMI (1 part in 50) for final dilution~~.	+	#* For "D: fMLP," add 20 µL of stock to 980 µL RPMI IL-3.
-	#* For "E: Anti-IgE," add 30 µL of ~~200 µL~~ stock to ~~2670~~ µL RPMI IL-3 (1 part in 90).	+	#* For "E: Anti-IgE," add 10 µL of stock to 890 µL RPMI IL-3 (1 part in 90).
	#* For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.		#* For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.
	#* For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.		#* For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
	#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.		#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
	#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.		#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
		+	#* For "P: Oxyplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
		+	#* For remainder of oxyplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
	# Transfer 250 µL of each stimulant into separate PP tubes, labeled A-O		# Transfer 250 µL of each stimulant into separate PP tubes, labeled A-O
	# Gently mix blood by inverting green top tube.		# Gently mix blood by inverting green top tube.
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	# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).		# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).
	# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:		# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:
-	#: To ~~940~~ µL staining buffer, add ~~140~~ μL each of:	+	#: To 1240 µL staining buffer, add 190 μL each of:
	#* CD63-FITC		#* CD63-FITC
	#* CD123-PC5		#* CD123-PC5
	#* HLA-DR-APC		#* HLA-DR-APC
-	#: and 70 µL each of:	+	#: and 95 µL each of:
	#* CD203c-PC7		#* CD203c-PC7
	#* CRTH2-PE		#* CRTH2-PE

Alex Ma: /* Procedure */

2010-12-07T02:44:38Z

Procedure

←Older revision		Revision as of 02:44, 7 December 2010
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	# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).		# Incubate tubes for 20 minutes in 37°C incubator (5% CO<sub>2</sub>).
	# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:		# While incubating, prepare the antibody cocktail and store in 4°C refrigerator until ready for use:
-	#* To ~~1 mL~~ staining buffer, add ~~150~~ μL each of:	+	#: To 940 µL staining buffer, add 140 μL each of:
	#* CD63-FITC		#* CD63-FITC
	#* CD123-PC5		#* CD123-PC5
	#* HLA-DR-APC		#* HLA-DR-APC
-	#: and 75 µL each of:	+	#: and 70 µL each of:
	#* CD203c-PC7		#* CD203c-PC7
	#* CRTH2-PE		#* CRTH2-PE
-	#: ~~'''Vortex'''~~	+	#: Mix by vortex or inversion (if made in PP tube with cap)
-	# After the 20 minutes of incubation, remove the samples.	+	# After the 20 minutes of incubation, remove the samples, putting them on ice.
-	~~# Put samples~~ on ice.	+	# Add 100 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.		# Gently mix cell suspension with mAb cocktail by flicking tube with finger or using #3 setting on vortex.
-	# Incubate for 20 min at 4°C~~. '''Protect from~~ light~~'''~~.	+	# Incubate for 30 min at 4°C, taking care to limit exposure to light.
-	# ~~Add~~ 2 mL staining buffer.	+	# After incubation, wash samples with 2 mL staining buffer per tube.
-	# Invert tubes 3X to mix	+	# Invert tubes (covering opening w/ parafilm) 3X to mix
	# Centrifuge for 5 minutes at 300g, RT		# 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 3 mL of 1x FACS lysing solution one tube at a time, ~~recap and '''invert repeatedly~~ until pellet is ~~fully~~ resuspended~~'''.~~	+	# Add 2 mL of 1x FACS lysing solution one tube at a time, inverting until pellet is resuspended (cover opening w/ parafilm)
-	# ~~Place~~ tubes in ~~the~~ dark for 15 minutes ~~or overnight in the refrigerator~~.	+	# Allow tubes to sit at RT (in dark) for 20 minutes. Following this, can suspend procedure by storing samples at 4ºC until ready to acquire flow data.
-	# ~~Top off tube with~~ staining buffer	+	# Add 1.5 mL staining buffer to each tube.
	# 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 50 μL staining buffer.	+	# Resuspend pellet in 75 μL staining buffer (final volume should be ~200 µL).
		+	# Transfer samples to new PS tubes for flow acquisition.
	# Aquire		# Aquire

Alex Ma: /* Procedure */

2010-12-07T02:35:31Z

Procedure

←Older revision		Revision as of 02:35, 7 December 2010
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	# Gently mix blood by inverting green top tube.		# Gently mix blood by inverting green top tube.
	# Transfer 250 μL of patient blood to each of the eleven PP tubes containing stimulants		# Transfer 250 μL of patient blood to each of the eleven PP tubes containing stimulants
-	# 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>).
-	# 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 ready for use:
-	#* To ~~900 uL~~ staining buffer, add 90 μL each of:	+	#* To 1 mL staining buffer, add 150 μL each of:
-	#* CD63	+	#* CD63-FITC
-	#* ~~CD203c~~	+	#* CD123-PC5
-	#* ~~CD123~~	+	#* HLA-DR-APC
-	#: and ~~12 μL~~ of:	+	#: and 75 µL each of:
-	#* ~~HLA~~-DR	+	#* CD203c-PC7
		+	#* CRTH2-PE
	#: '''Vortex'''		#: '''Vortex'''
	# After the 20 minutes of incubation, remove the samples.		# After the 20 minutes of incubation, remove the samples.

Alex Ma: /* Procedure */

2010-12-07T02:30:44Z

Procedure

Alex Ma: /* Procedure */

2010-12-07T02:19:39Z

Procedure

←Older revision		Revision as of 02:19, 7 December 2010
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	# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3		# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
	#* For "C: IL-3," use RPMI IL-3 as is.		#* For "C: IL-3," use RPMI IL-3 as is.
-	#* For "D: fMLP,"	+	#* For "D: fMLP," add 30 µL of 4 mM stock to 2370 µL RPMI IL-3 (1 part in 80), then add 20 µL of this dilution with 980 µL RPMI (1 part in 50) for final dilution.
		+	#* For "E: Anti-IgE," add 30 µL of 200 µL stock to 2670 µL RPMI IL-3 (1 part in 90).
		+	#* For "F: Carboplatin 1," add 30 µL of 20X carboplatin to 270 µL RPMI IL-3.
		+	#* For remainder of carboplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
		+	#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
		+	#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
	# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:
-	~~#* Add 270 μL of prepared Basophil medium to Drug 1 stimulant. '''Vortex'''~~
-	~~#* Transfer 270 μL of the Basophil medium prepared above to each of the eppendorf tubes labeled Drug 2-5.~~
-	~~#* Transfer 30 μL from Drug 1 stimulant into Drug 2 eppendorf, '''vortex'''. Repeat this from Drug 2 eppendorf into Drug 3 eppendorf~~
-	~~#* Continue making 10-fold dilutions in the same manner until all four Drug dilutions have been prepared (Drug 2 –Drug 5). '''Vortex''' after each dilution.~~
-	~~# 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 (drug allergen for tube 'G'; basophil medium for tube 'C', etc.).~~
	# Gently mix blood by inverting green top tube 4 times.		# 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.

Alex Ma: /* Procedure */

2010-12-06T22:42:25Z

Procedure

←Older revision		Revision as of 22:42, 6 December 2010
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	#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex		#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
	# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3		# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
-	# ~~Add 270 μL of warm~~ RPMI IL-3 ~~to the following stimulants~~: ~~anti-IgE and~~ fMLP, ~~Drug 1.~~	+	#* For "C: IL-3," use RPMI IL-3 as is.
		+	#* For "D: fMLP,"
	# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:
	#* Add 270 μL of prepared Basophil medium to Drug 1 stimulant. '''Vortex'''		#* Add 270 μL of prepared Basophil medium to Drug 1 stimulant. '''Vortex'''

Alex Ma: /* Procedure */

2010-12-06T22:39:41Z

Procedure

←Older revision		Revision as of 22:39, 6 December 2010
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	#Prepare RPMI w/IL-3 as follows:		#Prepare RPMI w/IL-3 as follows:
	#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex		#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
-	#*Prepare	+	# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
	# Add 270 μL of warm RPMI IL-3 to the following stimulants: anti-IgE and fMLP, Drug 1.		# Add 270 μL of warm RPMI IL-3 to the following stimulants: anti-IgE and fMLP, Drug 1.
	# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:

Alex Ma: /* Procedure */

2010-12-06T22:38:34Z

Procedure

←Older revision		Revision as of 22:38, 6 December 2010
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	#*N: Cisplatin 4 (0.001X)		#*N: Cisplatin 4 (0.001X)
	#*O: Cisplatin 5 (0.0001X)		#*O: Cisplatin 5 (0.0001X)
-	# Prepare RPMI w/IL-3 as follows ~~(set aside for step 10)~~:	+	#Prepare RPMI w/IL-3 as follows:
-	#* Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex	+	#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
		+	#*Prepare
	# Add 270 μL of warm RPMI IL-3 to the following stimulants: anti-IgE and fMLP, Drug 1.		# Add 270 μL of warm RPMI IL-3 to the following stimulants: anti-IgE and fMLP, Drug 1.
	# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:

Alex Ma: /* Procedure */

2010-12-06T22:37:45Z

Procedure

←Older revision		Revision as of 22:37, 6 December 2010
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	#*N: Cisplatin 4 (0.001X)		#*N: Cisplatin 4 (0.001X)
	#*O: Cisplatin 5 (0.0001X)		#*O: Cisplatin 5 (0.0001X)
-	# Prepare ~~Basophil medium (BM)~~ as follows (set aside for step 10):	+	# Prepare RPMI w/IL-3 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). ~~'''Vortex'''~~	+	#* Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
-	# Add 270 μL of warm BM to the following stimulants: anti-IgE and fMLP, Drug 1.	+	# Add 270 μL of warm RPMI IL-3 to the following stimulants: anti-IgE and fMLP, Drug 1.
	# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:		# Prepare 10-fold serial dilutions of the Drug 1 stimulant as follows:
	#* Add 270 μL of prepared Basophil medium to Drug 1 stimulant. '''Vortex'''		#* Add 270 μL of prepared Basophil medium to Drug 1 stimulant. '''Vortex'''

Alex Ma: /* Procedure */

2010-12-06T22:30:39Z

Procedure

←Older revision		Revision as of 22:30, 6 December 2010
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	# Remove pre-made stimulants (fMLP, Drug, Anti-IgE) from freezer and let thaw.		# Remove pre-made stimulants (fMLP, Drug, Anti-IgE) from freezer and let thaw.
	# Place 5 mL RPMI in water bath for ~5-10 minutes.		# Place 5 mL RPMI in water bath for ~5-10 minutes.
-	# ~~Label four eppendorf tubes for Drug 2-5, and nine~~ 5 mL PP tubes for conditions (A-I).	+	# Prepare 15 5 mL PP tubes for conditions (A-O):
		+	#*A: Unstained
		+	#*B: RPMI
		+	#*C: IL-3
		+	#*D: fMLP
		+	#*E: Anti-IgE
		+	#*F: Carboplatin 1 (1X)
		+	#*G: Carboplatin 2 (0.1X)
		+	#*H: Carboplatin 3 (0.01X)
		+	#*I: Carboplatin 4 (0.001X)
		+	#*J: Carboplatin 5 (0.0001X)
		+	#*K: Cisplatin 1 (1X)
		+	#*L: Cisplatin 2 (0.1X)
		+	#*M: Cisplatin 3 (0.01X)
		+	#*N: Cisplatin 4 (0.001X)
		+	#*O: Cisplatin 5 (0.0001X)
	# Prepare Basophil medium (BM) as follows (set aside for step 10):		# Prepare Basophil medium (BM) 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). '''Vortex'''		#* Add 4 μL of IL-3 into 2.0 mL of warm RPMI (use 5 mL PP tube). '''Vortex'''

←Older revision		Revision as of 02:30, 7 December 2010
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	#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex		#*Add 4 μL of aliquot IL-3 (10 µL/mL) into 2.0 mL of warm RPMI in 5 mL PP tube. Mix well by inverting or vortex
	# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3		# Except for "Unstained" and "RPMI" (tubes A and B), prepare all stimulants using RPMI IL-3
		+	#* For "A: Unstained" and "B: RPMI," use RPMI (no IL-3)
	#* For "C: IL-3," use RPMI IL-3 as is.		#* For "C: IL-3," use RPMI IL-3 as is.
	#* For "D: fMLP," add 30 µL of 4 mM stock to 2370 µL RPMI IL-3 (1 part in 80), then add 20 µL of this dilution with 980 µL RPMI (1 part in 50) for final dilution.		#* For "D: fMLP," add 30 µL of 4 mM stock to 2370 µL RPMI IL-3 (1 part in 80), then add 20 µL of this dilution with 980 µL RPMI (1 part in 50) for final dilution.
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	#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.		#* For "K: Cisplatin 1," add 30 µL of 20X cisplatin to 270 µL RPMI IL-3.
	#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.		#* For remainder of cisplatin series, take 30 µL from previous concentration in series and mix with 270 µL RPMI IL-3.
-	# ~~Prepare 10-fold serial dilutions~~ of ~~the Drug 1~~ stimulant ~~as follows:~~	+	# Transfer 250 µL of each stimulant into separate PP tubes, labeled A-O
-	# Gently mix blood by inverting green top tube ~~4 times~~.	+	# Gently mix blood by inverting green top tube.
-	# ~~'''IN THE HOOD''', add~~ 250 μL of patient blood to ~~all~~ eleven tubes.	+	# Transfer 250 μL of patient blood to each of the eleven PP tubes containing stimulants
	# 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: