View source for DsRNA Mediated Knockdown of S2 Cells

==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
<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:Knockdown]]
[[Category:Drosophila]]