GHV-68 Plaque Assay - Revision history

Torsten Waldminghaus at 11:12, 19 February 2009

Torsten Waldminghaus — Thu, 19 Feb 2009 11:12:07 GMT

←Older revision		Revision as of 11:12, 19 February 2009
Line 74:		Line 74:
	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

-	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. ~~-->~~	+	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information.
	[[Category:Protocol]]		[[Category:Protocol]]
-	[[Category:Needs attention]] ~~<!--Delete this line once the protocol is complete-->~~	+	[[Category:Needs attention]]
-		+
-	~~<!-- Move the relevant categories above this line to tag your protocol with the label~~	+
	[[Category:In vitro]]		[[Category:In vitro]]

Stevej: /* Overview */

Stevej — Wed, 06 Jun 2007 20:49:55 GMT

Overview

←Older revision		Revision as of 20:49, 6 June 2007
Line 1:		Line 1:
	==Overview==		==Overview==

-	Plaque assay for gHV-68 using 3T12 fibroblasts~~. Stolen from Virgin lab protocols~~.	+	Plaque assay for gHV-68 using 3T12 fibroblasts.

	==Materials==		==Materials==

Stevej: /* Procedure */

Stevej — Mon, 04 Jun 2007 17:49:00 GMT

Procedure

←Older revision		Revision as of 17:49, 4 June 2007
Line 48:		Line 48:
	Day 7:		Day 7:

-	#Add neutral red (dilute the lab stock 2-3/100 in PBS and filter before using) approximately 1-2 ml/well, distributed over the surface of the methylcellulose. This will diffuse to cover the monolayer and allow visualization of the plaques.	+	#Add [[neutral red]] (dilute the lab stock 2-3/100 in PBS and filter before using) approximately 1-2 ml/well, distributed over the surface of the methylcellulose. This will diffuse to cover the monolayer and allow visualization of the plaques.

	Day 8		Day 8

Stevej: /* References */

Stevej — Mon, 04 Jun 2007 17:41:30 GMT

References

←Older revision		Revision as of 17:41, 4 June 2007
Line 66:		Line 66:
	<!-- 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>
-	~~#Goldbeter-PNAS-1981 pmid=6947258~~	+
-	~~#Jacob-JMB-1961 pmid=13718526~~	+
-	~~#Ptashne-Genetic-Switch isbn=0879697164~~	+
	</biblio>		</biblio>

Stevej: /* Notes */

Stevej — Mon, 04 Jun 2007 17:41:02 GMT

Notes

←Older revision		Revision as of 17:41, 4 June 2007
Line 58:		Line 58:
	==Notes==		==Notes==
	#List troubleshooting tips here.		#List troubleshooting tips here.
-	~~#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.~~	+
-	~~#Anecdotal observations that might be of use to others can also be posted here.~~	+

	Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

Stevej: /* Procedure */

Stevej — Mon, 04 Jun 2007 17:39:00 GMT

Procedure

←Older revision		Revision as of 17:39, 4 June 2007
Line 45:		Line 45:

	#Incubate 37C, 5% CO2 for 6 d.		#Incubate 37C, 5% CO2 for 6 d.
		+
		+	Day 7:
		+
		+	#Add neutral red (dilute the lab stock 2-3/100 in PBS and filter before using) approximately 1-2 ml/well, distributed over the surface of the methylcellulose. This will diffuse to cover the monolayer and allow visualization of the plaques.
		+
		+	Day 8
		+
		+	#Count plaques. The neutral red is toxic to the 3T12 cells, so the plaques should be read less than 24 h after addition of the dye.
		+
		+	#Tape and double bag the plates and autoclave.

	==Notes==		==Notes==

Stevej: /* Procedure */

Stevej — Wed, 30 May 2007 19:46:56 GMT

Procedure

←Older revision		Revision as of 19:46, 30 May 2007
Line 34:		Line 34:
	##Discard media from 6-well plates into a beaker in the hood.		##Discard media from 6-well plates into a beaker in the hood.

-	##Transfer 200 ul from each of the wells in the dilution matrix into one well of a 6-well plate. Pipette the sample down the wall of the well of the 6-well plate; don't squirt it into the middle of the monolayer of cells, since this can make a false plaque. Start at the highest dilution (lowest concentration of virus) in the 96-well plate and move up a row to more and more concentrated virus using the same tip. One must change tips, obviously, when moving to a new virus sample (new column).	+	#Transfer 200 ul from each of the wells in the dilution matrix into one well of a 6-well plate. Pipette the sample down the wall of the well of the 6-well plate; don't squirt it into the middle of the monolayer of cells, since this can make a false plaque. Start at the highest dilution (lowest concentration of virus) in the 96-well plate and move up a row to more and more concentrated virus using the same tip. One must change tips, obviously, when moving to a new virus sample (new column).

-	##Optional: Add an additional 200 ul/well of D-10 to each well for a total of 400 ul/well. This extra volume prevents drying of the monolayer during the adsorption step.	+	#Optional: Add an additional 200 ul/well of D-10 to each well for a total of 400 ul/well. This extra volume prevents drying of the monolayer during the adsorption step.

-	##Adsorb the virus at 37C for 1-2 hrs; use frequent rocking (every 10-15 min by hand) to avoid drying the cells. Note: Previous protocols have done the dilutions in eppendorf tubes and the adsorption step with 500 ul per well and rocked the plates on the automatic rocker at RT. I've had problems with the automatic rocker and the lower volume (400 ul/well) that is suggested in this protocol. I think the rocker may actually promote drying of the center of the well by drawing the fluid to the side of the well via capillary action.	+	#Adsorb the virus at 37C for 1-2 hrs; use frequent rocking (every 10-15 min by hand) to avoid drying the cells. Note: Previous protocols have done the dilutions in eppendorf tubes and the adsorption step with 500 ul per well and rocked the plates on the automatic rocker at RT. I've had problems with the automatic rocker and the lower volume (400 ul/well) that is suggested in this protocol. I think the rocker may actually promote drying of the center of the well by drawing the fluid to the side of the well via capillary action.

-	##Overlay approx 5 ml warm methylcellulose per well.	+	#Overlay approx 5 ml warm methylcellulose per well.

-	##Hand rock each plate gently to mix the virus-containing adsorption media with the media in the methylcellulose. This is in an attempt to avoid pockets of virus forming between the layer of cells and the methylcellulose.	+	#Hand rock each plate gently to mix the virus-containing adsorption media with the media in the methylcellulose. This is in an attempt to avoid pockets of virus forming between the layer of cells and the methylcellulose.

-	##Incubate 37C, 5% CO2 for 6 d.	+	#Incubate 37C, 5% CO2 for 6 d.

	==Notes==		==Notes==

Stevej: /* Procedure */

Stevej — Wed, 30 May 2007 19:46:11 GMT

Procedure

←Older revision		Revision as of 19:46, 30 May 2007
Line 32:		Line 32:
	##Change tips.		##Change tips.
	##Repeat for row B to row C, etc.		##Repeat for row B to row C, etc.
		+	##Discard media from 6-well plates into a beaker in the hood.
		+
		+	##Transfer 200 ul from each of the wells in the dilution matrix into one well of a 6-well plate. Pipette the sample down the wall of the well of the 6-well plate; don't squirt it into the middle of the monolayer of cells, since this can make a false plaque. Start at the highest dilution (lowest concentration of virus) in the 96-well plate and move up a row to more and more concentrated virus using the same tip. One must change tips, obviously, when moving to a new virus sample (new column).
		+
		+	##Optional: Add an additional 200 ul/well of D-10 to each well for a total of 400 ul/well. This extra volume prevents drying of the monolayer during the adsorption step.
		+
		+	##Adsorb the virus at 37C for 1-2 hrs; use frequent rocking (every 10-15 min by hand) to avoid drying the cells. Note: Previous protocols have done the dilutions in eppendorf tubes and the adsorption step with 500 ul per well and rocked the plates on the automatic rocker at RT. I've had problems with the automatic rocker and the lower volume (400 ul/well) that is suggested in this protocol. I think the rocker may actually promote drying of the center of the well by drawing the fluid to the side of the well via capillary action.
		+
		+	##Overlay approx 5 ml warm methylcellulose per well.
		+
		+	##Hand rock each plate gently to mix the virus-containing adsorption media with the media in the methylcellulose. This is in an attempt to avoid pockets of virus forming between the layer of cells and the methylcellulose.
		+
		+	##Incubate 37C, 5% CO2 for 6 d.

	==Notes==		==Notes==

Stevej: /* Procedure */

Stevej — Wed, 30 May 2007 19:45:06 GMT

Procedure

←Older revision		Revision as of 19:45, 30 May 2007
Line 25:		Line 25:
	##Each column represents a different sample of virus to be diluted.		##Each column represents a different sample of virus to be diluted.
	##Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.		##Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.
-	*Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.	+	###Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.
	##Aliquot 225 ul/well fresh media in row B-H.		##Aliquot 225 ul/well fresh media in row B-H.
	##Aliquot 250 ul/well neat virus in row A.		##Aliquot 250 ul/well neat virus in row A.

Stevej: /* Procedure */

Stevej — Wed, 30 May 2007 19:44:47 GMT

Procedure

←Older revision		Revision as of 19:44, 30 May 2007
Line 25:		Line 25:
	##Each column represents a different sample of virus to be diluted.		##Each column represents a different sample of virus to be diluted.
	##Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.		##Each row represents a 10-fold dilution of the virus, ie—row A is 100, row B is 10-1, row C is 10-2, etc.
-	::*Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.	+	*Don't forget to include a mock (media only) and a stock viral solution (previously titered) as controls.
	##Aliquot 225 ul/well fresh media in row B-H.		##Aliquot 225 ul/well fresh media in row B-H.
	##Aliquot 250 ul/well neat virus in row A.		##Aliquot 250 ul/well neat virus in row A.