DsRNA Mediated Knockdown of S2 Cells: Difference between revisions

Revision as of 20:05, 29 September 2009

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.

@@ Line 10: / Line 10: @@
 **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
+**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:Knockdown]]
 [[Category:Drosophila]]