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

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.