# Altman:Protocols/Protein Purification/SF9 transfection

(Difference between revisions)
 Revision as of 13:26, 19 February 2013 (view source)← Previous diff Current revision (14:38, 19 February 2013) (view source) (3 intermediate revisions not shown.) Line 13: Line 13: : PROTOCOL FOR COUNTING CELLS: : PROTOCOL FOR COUNTING CELLS: : 1) Get SF9 cells from the incubator shaker, and put in TC hood : 1) Get SF9 cells from the incubator shaker, and put in TC hood - : 2) Remove 10 μL of the cells, being sure to shake the flask beforehand + : 2) Remove 10 uL of the cells, being sure to shake the flask beforehand - : 3) Dilute the cells 5x with 40 \uL of Trypan blue + : 3) Dilute the cells 5x with 40 uL of Trypan blue - : 4) Place 10 μL in a hemocytometer (from Shirley’s bench) + : 4) Place 10 uL in a hemocytometer (from Shirley’s bench) : 5) Count the cells in 4 square grids : 5) Count the cells in 4 square grids - :: 1 grid = 10-4 mL + :: 1 grid = 10^-4 mL - : Mean # of total / dead cells per grid = + :: Cell Density = (mean # of cells per grid)*5*10^4 cells/mL - : Viability of cells = + - : Cell Density = (mean # of cells per grid)*5*10^4 cells/mL = + - 2.Determine the volume of cells required for the day + 2. Determine the volume of cells required for the day : Each transfection requires 15 mL of SF9 cells at 1*10^6 cells/mL, or 1.5*10^7 cells : Each transfection requires 15 mL of SF9 cells at 1*10^6 cells/mL, or 1.5*10^7 cells - 3. Wash the cells by spinning 2 min at 500 rpm in a 15 mL conical in a Beckman TJ-6 centrifuge + 3. Wash the cells by spinning 10 min at 500 rpm in a 15 mL conical in a Beckman TJ-6 centrifuge - 4. Remove the supernatant, and re-suspend the pellet in unsupplemented SF 900 II media to a cell density of ~1.5*10^6 cells/mL (10 mL of media per transfection) + 4. Remove the supernatant, and re-suspend the pellet in unsupplemented SF 900 II media to a cell density of 1.5*10^6 cells/mL (10 mL of media per transfection) 5. Replace cells in 28° shaker until the complexes are ready for transfection 5. Replace cells in 28° shaker until the complexes are ready for transfection - 6. Dilute ~10 \ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube + 6. Dilute 10 ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube - 6. Dilute 150 μl of ESCORT IV cationic lipids in 1.35 mL of unsupplemented media; mix by inverting tube + - 7. Add diluted DNA dropwise to diluted lipids, mixing while adding.  Ensure complete mixing by inverting tube afterwards.  Allow mixture to sit in the hood for at least 15’ to promote complex formation. + - 8. Meanwhile, retrieve and count unsupplemented cells; dilute the cells to a final density of 1.25 *10^6 cells/mL in unsupplemented media. + - Mean # of total / dead cells per grid = + 7. Dilute 150 ul of ESCORT IV cationic lipids in 1.35 mL of unsupplemented media; mix by inverting tube - Viability of cells = + - Cell Density = (mean # of cells per grid)*5*10^4 cells/mL = + 8. Add diluted DNA dropwise to diluted lipids, mixing while adding - 9. Dispense 12 mL of cell culture into a plastic 125 mL culture flask (disposable flask with a vented top; 0.2 μm pore) + : Ensure complete mixing by inverting tube afterwards.  Allow mixture to sit in the hood for at least 15’ to promote complex formation. - 10. Add 3 mL DNA:lipid complexes to the cell culture. + - 11. Replace cell culture in 28° shaker; cells may be harvested in 48-72 hours. + 9. Meanwhile, retrieve and count  cells; dilute the cells to a final density of 1.25 *10^6 cells/mL in unsupplemented media + + 10. Dispense 12 mL of cell culture into a plastic 125 mL culture flask + + 11. Add 3 mL DNA:lipid complexes to the cell culture. + + 12. Replace cell culture in 28° shaker; cells may be harvested in 48-72 hours

## Current revision

Department of Physics, Willamette University

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## Direct transfection of SF9 cells for small-scale protein purification

(Protocol is from DA, based on ZB, based on Novagen protocol)

• This protocol is for transfecting 15 mL cultures of SF9 cells at 1*10^6 cells/mL. 3 mL of DNA:lipid complexes are used for each 15 mL culture of cells. The protocol can be scaled up slightly by making a larger batch of DNA:lipid complexes (I have made up to 10 mL at a time) and splitting it among multiple 15 mL cultures in separate flasks.

1. Determine the cell density

PROTOCOL FOR COUNTING CELLS:
1) Get SF9 cells from the incubator shaker, and put in TC hood
2) Remove 10 uL of the cells, being sure to shake the flask beforehand
3) Dilute the cells 5x with 40 uL of Trypan blue
4) Place 10 uL in a hemocytometer (from Shirley’s bench)
5) Count the cells in 4 square grids
1 grid = 10^-4 mL
Cell Density = (mean # of cells per grid)*5*10^4 cells/mL

2. Determine the volume of cells required for the day

Each transfection requires 15 mL of SF9 cells at 1*10^6 cells/mL, or 1.5*10^7 cells

3. Wash the cells by spinning 10 min at 500 rpm in a 15 mL conical in a Beckman TJ-6 centrifuge

4. Remove the supernatant, and re-suspend the pellet in unsupplemented SF 900 II media to a cell density of 1.5*10^6 cells/mL (10 mL of media per transfection)

5. Replace cells in 28° shaker until the complexes are ready for transfection

6. Dilute 10 ug of plasmid DNA in 1.5 mL unsupplemented media; mix by inverting tube

7. Dilute 150 ul of ESCORT IV cationic lipids in 1.35 mL of unsupplemented media; mix by inverting tube