Griffin:Puromycin Selection - Revision history

Korey Griffin: /* Measuring Puromycin cytotoxicity */

Korey Griffin — Tue, 09 Mar 2010 18:27:27 GMT

Measuring Puromycin cytotoxicity

←Older revision		Revision as of 18:27, 9 March 2010
Line 41:		Line 41:
	*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.		*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.

-	==Measuring Puromycin ~~cytotoxicity~~==	+	==Measuring Puromycin cytotoxic==

	The measurement of cell viability and growth is an important factor in titrating antibiotic. Several approaches exist;		The measurement of cell viability and growth is an important factor in titrating antibiotic. Several approaches exist;

Korey Griffin at 18:27, 9 March 2010

Korey Griffin — Tue, 09 Mar 2010 18:27:07 GMT

←Older revision		Revision as of 18:27, 9 March 2010
Line 7:		Line 7:
	Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition. Media should be changed out every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.		Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition. Media should be changed out every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.

-	==Puromycin Selection (shRNA ~~protocol~~) ~~for selection of stably transfected cells~~==	+	==Puromycin Selection post transfection (shRNA)/transduction (lenti)==

	*48 hours post-[http://openwetware.org/wiki/Griffin:shRNA_Transfection shRNA transfection], aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.		*48 hours post-[http://openwetware.org/wiki/Griffin:shRNA_Transfection shRNA transfection], aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
Line 25:		Line 25:
	*Jurkat: 4		*Jurkat: 4

-	==Titrating puromycin ~~for selecting transfected cell lines~~==	+	==Titrating puromycin==

	*Plate 2 x 105 cells in each well of a 6-well plate containing 3 ml of the appropriate complete medium plus increasing concentrations of puromycin (i.e., 0, 1.0, 2.5, 5.0, 7.5, and 10.0 µg/ml)		*Plate 2 x 105 cells in each well of a 6-well plate containing 3 ml of the appropriate complete medium plus increasing concentrations of puromycin (i.e., 0, 1.0, 2.5, 5.0, 7.5, and 10.0 µg/ml)
Line 40:		Line 40:
	*'''High protein expression levels''' – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.		*'''High protein expression levels''' – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
	*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.		*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
		+
		+	==Measuring Puromycin cytotoxicity==
		+
		+	The measurement of cell viability and growth is an important factor in titrating antibiotic. Several approaches exist;
		+
		+	*[http://en.wikipedia.org/wiki/Trypan_blue Trypan blue]; live cells or tissues with intact cell membranes are not colored. Since cells are selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed; however, it traverses the membrane in a dead cell.
		+	*Bright field microscope can indicate adherent cells exhibiting loss of plate attachment
		+	*[http://en.wikipedia.org/wiki/MTT_assay MTS assay] water-soluble formazan product with absorbance maximum at 490-500 nm in PBS takes place only when reductase enzymes are active, and therefore conversion is a function of viable (living) cells.

	==References==		==References==

Linda Kippner: added Jurkat cell puromycin concentration based on own experimental data

Linda Kippner — Fri, 13 Mar 2009 20:13:22 GMT

added Jurkat cell puromycin concentration based on own experimental data

←Older revision		Revision as of 20:13, 13 March 2009
Line 23:		Line 23:
	*HEK293: 2		*HEK293: 2
	*A431: 1		*A431: 1
		+	*Jurkat: 4

	==Titrating puromycin for selecting transfected cell lines==		==Titrating puromycin for selecting transfected cell lines==

Korey Griffin: /* Puromycin Selection (shRNA protocol) for selection of stably transfected cells */

Korey Griffin — Thu, 18 Dec 2008 22:18:09 GMT

Puromycin Selection (shRNA protocol) for selection of stably transfected cells

←Older revision		Revision as of 22:18, 18 December 2008
Line 17:		Line 17:
	Suggested working conditions for selection in some mammalian cells:		Suggested working conditions for selection in some mammalian cells:

-	*Hela human uterus: 3 µg/ml	+	*Hela human uterus: 3 µg/ml
-	*HEK293 human embryonic kidney: 3	+	*HEK293 human embryonic kidney: 3
-	*B16 mouse melanoma: 1-3	+	*B16 mouse melanoma: 1-3
	*PC1.0 hamster adenocarcinoma: 10		*PC1.0 hamster adenocarcinoma: 10
		+	*HEK293: 2
		+	*A431: 1

	==Titrating puromycin for selecting transfected cell lines==		==Titrating puromycin for selecting transfected cell lines==

Korey Griffin: /* Puromycin Selection (shRNA protocol) for selection of stably transfected cells */

Korey Griffin — Thu, 30 Oct 2008 22:50:53 GMT

Puromycin Selection (shRNA protocol) for selection of stably transfected cells

←Older revision		Revision as of 22:50, 30 October 2008
Line 9:		Line 9:
	==Puromycin Selection (shRNA protocol) for selection of stably transfected cells==		==Puromycin Selection (shRNA protocol) for selection of stably transfected cells==

-	*48 hours post-[http://openwetware.org/wiki/Griffin:shRNA_Transfection shRNA ~~plasmid~~ transfection], aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.	+	*48 hours post-[http://openwetware.org/wiki/Griffin:shRNA_Transfection shRNA transfection], aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
	*Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.		*Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
	*Monitor the cells daily. Puromycin selection requires a minimum of 48 hours.		*Monitor the cells daily. Puromycin selection requires a minimum of 48 hours.

Korey Griffin: /* Factors Influencing Successful Transfection */

Korey Griffin — Thu, 30 Oct 2008 22:50:29 GMT

Factors Influencing Successful Transfection

←Older revision		Revision as of 22:50, 30 October 2008
Line 33:		Line 33:

	*'''Concentration and purity of nucleic acids''' – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.		*'''Concentration and purity of nucleic acids''' – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
-	*'''Transfection in serum-free media''' – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is	+	*'''Transfection in serum-free media''' – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is observed.
-	observed.	+
	*'''No antibiotics in transfection medium''' – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.		*'''No antibiotics in transfection medium''' – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.
	*'''High protein expression levels''' – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.		*'''High protein expression levels''' – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
-	*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest	+	*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
-	transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.	+

	==References==		==References==

Korey Griffin: /* Factors Influencing Successful Transfection */

Korey Griffin — Thu, 30 Oct 2008 22:50:05 GMT

Factors Influencing Successful Transfection

←Older revision		Revision as of 22:50, 30 October 2008
Line 32:		Line 32:
	==Factors Influencing Successful Transfection==		==Factors Influencing Successful Transfection==

-	1. Concentration and purity of nucleic acids – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.	+	*'''Concentration and purity of nucleic acids''' – Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
-		+	*'''Transfection in serum-free media''' – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is
-	2. Transfection in serum-free media – the highest transfection efficiencies can be obtained if the cells are exposed to the transfection complexes in serum free conditions followed by the addition of medium containing twice the amount of normal serum to the complex medium 3–5 hrs post transfection (leaving the complexes on the cells). However, the transfection medium can be replaced with normal growth medium if high toxicity is	+
	observed.		observed.
-		+	*'''No antibiotics in transfection medium''' – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.
-	3. No antibiotics in transfection medium – The presence of antibiotics can adversely affect the transfection efficiency and lead to increased toxicity levels in some cell types. It is recommended that these additives be initially excluded until optimized conditions are achieved, then these components can be added, and the cells can be monitored for any changes in the transfection results.	+	*'''High protein expression levels''' – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.
-		+	*'''Cell history, density, and passage number''' –It is very important to use healthy cells that are regularly passaged and in growth phase. The highest
-	4. High protein expression levels – Some proteins when expressed at high levels can by cytotoxic; this effect can also be cell line specific.	+	transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.
-		+
-	5. Cell history, density, and passage ~~number–It~~ is very important to use healthy cells that are regularly passaged and in growth phase. The highest	+
-	transfection efficiencies are achieved if cells are plated the day before. However, adequate time should be allowed to allow the cells to recover from the passaging (generally >12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results.	+

	==References==		==References==

Korey Griffin: /* References */

Korey Griffin — Thu, 30 Oct 2008 22:49:02 GMT

References

←Older revision		Revision as of 22:49, 30 October 2008
Line 50:		Line 50:
	[http://www.openbiosystems.com/ Open Biosystems]		[http://www.openbiosystems.com/ Open Biosystems]

-	~~Murnane, J. P., Yezzi, M. J., Young, B. R. (1990) Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Res, 18: 2733-2738.~~	+	<biblio>
-		+	#Paper1 pmid=2339059
-	~~Haber, J. E. (1999) DNA repair. Gatekeepers of recombination. Nature, 398: 665-667.~~	+	#Paper2 pmid=10227286
-		+	#Paper3 pmid=15761468
-	~~Glover, D. J., Lipps, H. J., Jans, D. A. (2005). Towards safe, non-viral therapeutic gene expression in humans. Nature Rev Gen, 6: 299-310.~~	+	</biblio>

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]

Korey Griffin: /* References */

Korey Griffin — Thu, 30 Oct 2008 22:38:53 GMT

References

←Older revision		Revision as of 22:38, 30 October 2008
Line 45:		Line 45:

	==References==		==References==
		+
		+	[http://www.amaxa.com/stable-transfection.html Amaxa]

	[http://www.openbiosystems.com/ Open Biosystems]		[http://www.openbiosystems.com/ Open Biosystems]

Korey Griffin at 22:38, 30 October 2008

Korey Griffin — Thu, 30 Oct 2008 22:38:17 GMT

←Older revision		Revision as of 22:38, 30 October 2008
Line 3:		Line 3:
	[[Image:puromycin.gif]]		[[Image:puromycin.gif]]

-	Puromycin dihydrochloride is a protein synthesis ~~inhibitor~~. ~~This aminonuclease antibiotic~~ used for selection and maintenance of cell lines expressing a transfected pac gene (S. alboniger), whose product, puromycin acetyltransferase, inactivates puromycin via acetylation; recommended concentration in cell culture 1-10µg/ml. Lot-to-lot variations in potency exist for all selection antibiotics, each new lot of puromycin should be titrated. The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.	+	Puromycin dihydrochloride is a aminonuclease antibiotic that inhibits protein synthesis. Puromycin is used for selection and maintenance of cell lines expressing a transfected pac gene (S. alboniger), whose product, puromycin acetyltransferase, inactivates puromycin via acetylation; recommended concentration in cell culture 1-10µg/ml. Lot-to-lot variations in potency exist for all selection antibiotics, each new lot of puromycin should be titrated. The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
		+
		+	Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition. Media should be changed out every 2-3 days. This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.

	==Puromycin Selection (shRNA protocol) for selection of stably transfected cells==		==Puromycin Selection (shRNA protocol) for selection of stably transfected cells==
Line 45:		Line 47:

	[http://www.openbiosystems.com/ Open Biosystems]		[http://www.openbiosystems.com/ Open Biosystems]
		+
		+	Murnane, J. P., Yezzi, M. J., Young, B. R. (1990) Recombination events during integration of transfected DNA into normal human cells. Nucleic Acids Res, 18: 2733-2738.
		+
		+	Haber, J. E. (1999) DNA repair. Gatekeepers of recombination. Nature, 398: 665-667.
		+
		+	Glover, D. J., Lipps, H. J., Jans, D. A. (2005). Towards safe, non-viral therapeutic gene expression in humans. Nature Rev Gen, 6: 299-310.

	[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:Mammalian cell culture]]