DsRNA Mediated Knockdown of S2 Cells - Revision history

Davebrid at 20:14, 29 September 2009

2009-09-29T20:14:17Z

← Older revision		Revision as of 20:14, 29 September 2009
Line 12:		Line 12:
	**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row		**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row
	<pre>x * 4 (rows per square) * 10^5 (dilution factor) = concentration of cells/mL</pre>		<pre>x * 4 (rows per square) * 10^5 (dilution factor) = concentration of cells/mL</pre>
	**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.		*Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		*Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
	**Refeed cells with fresh media and dsRNA daily, typically for 4 days.		*Refeed cells with fresh media and dsRNA daily, typically for 4 days.

	~~{{math\|<VAR>i</VAR>}}~~

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid at 20:13, 29 September 2009

2009-09-29T20:13:55Z

← Older revision		Revision as of 20:13, 29 September 2009
Line 15:		Line 15:
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
	**Refeed cells with fresh media and dsRNA daily, typically for 4 days.		**Refeed cells with fresh media and dsRNA daily, typically for 4 days.

			{{math\|<VAR>i</VAR>}}

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid at 20:11, 29 September 2009

2009-09-29T20:11:25Z

← Older revision		Revision as of 20:11, 29 September 2009
Line 15:		Line 15:
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
	**Refeed cells with fresh media and dsRNA daily, typically for 4 days.		**Refeed cells with fresh media and dsRNA daily, typically for 4 days.

	~~<math>\text {abcdefghijklmnopqrstuvwxyzàáâãäåæçèéêëìíîïðñòóôõö÷øùúûüýþÿ}</math>~~

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid at 20:11, 29 September 2009

2009-09-29T20:11:13Z

← Older revision		Revision as of 20:11, 29 September 2009
Line 15:		Line 15:
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
	**Refeed cells with fresh media and dsRNA daily, typically for 4 days.		**Refeed cells with fresh media and dsRNA daily, typically for 4 days.

			<math>\text {abcdefghijklmnopqrstuvwxyzàáâãäåæçèéêëìíîïðñòóôõö÷øùúûüýþÿ}</math>

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid at 20:07, 29 September 2009

2009-09-29T20:07:28Z

← Older revision		Revision as of 20:07, 29 September 2009
Line 11:		Line 11:
	**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.		**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.
	**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row		**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row
	<pre>x * 4 (rows per square) * 10^5</pre>		<pre>x * 4 (rows per square) * 10^5 (dilution factor) = concentration of cells/mL</pre>
	**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.		**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA

Davebrid at 20:07, 29 September 2009

2009-09-29T20:07:08Z

← Older revision		Revision as of 20:07, 29 September 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*S2 Cells. See [[Culturing S2 Cells]]		*S2 Cells. See [[Culturing S2 Cells]]
	*dsRNA. See [[~~Preparation of~~ dsRNA]]		*dsRNA. See [[Designing and Preparing dsRNA]]
	*S2 Cell Media. See [[Culturing of S2 Cells]]		*S2 Cell Media. See [[Culturing S2 Cells]]

	==Protocol==		==Protocol==
Line 11:		Line 11:
	**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.		**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.
	**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row		**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row
	x * 4 (rows per square) * 10^5		<pre>x * 4 (rows per square) * 10^5</pre>
	**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.		**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.
	**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA		**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA

Davebrid at 20:05, 29 September 2009

2009-09-29T20:05:45Z

← Older revision		Revision as of 20:05, 29 September 2009
Line 10:		Line 10:
	**Add ~100 uL cells under coverslip to hemocytometer.		**Add ~100 uL cells under coverslip to hemocytometer.
	**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.		**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.
	**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where ~~\alpha~~ is number of cells per row		**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where x is number of cells per row
	~~{{math\|<var>&alpha</var>~~ x 4 (rows per square) x 10^5}}		x * 4 (rows per square) * 10^5
			**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.
			**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
			**Refeed cells with fresh media and dsRNA daily, typically for 4 days.

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid at 20:03, 29 September 2009

2009-09-29T20:03:25Z

← Older revision		Revision as of 20:03, 29 September 2009
Line 11:		Line 11:
	**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.		**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.
	**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where \alpha is number of cells per row		**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where \alpha is number of cells per row
	<~~math~~>\alpha~~\times~~ 4 (rows per square) ~~\times~~ 10^5 ~~(dilution factor)</math>~~		{{math\|<var>&alpha</var> x 4 (rows per square) x 10^5}}

	[[Category:Cell Culture]]		[[Category:Cell Culture]]
	[[Category:Knockdown]]		[[Category:Knockdown]]
	[[Category:Drosophila]]		[[Category:Drosophila]]

Davebrid: initial page

2009-09-29T20:00:46Z

initial page

New page

==Materials==
*S2 Cells. See [[Culturing S2 Cells]]
*dsRNA. See [[Preparation of dsRNA]]
*S2 Cell Media. See [[Culturing of S2 Cells]]

==Protocol==
*Take a near-confluent plate of S2 cells aspirate media and scrape into 1 mL of fresh media.
*Add 9 mL of fresh media and pipet to mix.
*Using hemocytometer count cells.
**Add ~100 uL cells under coverslip to hemocytometer.
**Count one row of 4 squares. For cells touching the edges of the square, only count the top and left line to avoid counting the same cells twice.
**Record the number of cells (should be 30-150 cells per row) and Calculate cell concentration, where \alpha is number of cells per row
<math>\alpha\times 4 (rows per square) \times 10^5 (dilution factor)</math>

[[Category:Cell Culture]]
[[Category:Knockdown]]
[[Category:Drosophila]]