DsRNA Mediated Knockdown of S2 Cells: Difference between revisions
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==Materials== | ==Materials== | ||
*S2 Cells. See [[Culturing S2 Cells]] | *S2 Cells. See [[Culturing S2 Cells]] | ||
*dsRNA. See [[ | *dsRNA. See [[Designing and Preparing dsRNA]] | ||
*S2 Cell Media. See [[Culturing | *S2 Cell Media. See [[Culturing S2 Cells]] | ||
==Protocol== | ==Protocol== | ||
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**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 | **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> | ||
*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]] | ||
Latest revision as of 20:14, 29 September 2009
Materials
- S2 Cells. See Culturing S2 Cells
- dsRNA. See Designing and Preparing dsRNA
- S2 Cell Media. See Culturing 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 x is number of cells per row
x * 4 (rows per square) * 10^5 (dilution factor) = concentration of cells/mL
- 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.