Difference between revisions of "DsRNA Mediated Knockdown of S2 Cells"

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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 \alpha is number of cells per row
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**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}}
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x * 4 (rows per square) * 10^5
 +
**Dilute cells to 1^6 cells per mL and seed out in 0.5mL per 12 well.
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**Let cells attach for >30 min, then add 0.5mL fresh media with 10 ug/mL dsRNA
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**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]]

Revision as of 20:05, 29 September 2009

Materials

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

    • 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.