Altman:Protocols/Protein Purification/SF9 transfection: Difference between revisions

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1. Determine the cell density  
1. Determine the cell density  
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 100 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 Trypan+PBS (Trypan+PBS = 1:1 Trypan blue + PBS)
: 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
grid Number of cells Number of dead cells
:: Cell Density = (mean # of cells per grid)*5*10^4 cells/mL  
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2
3
4
Mean # of total / dead cells per grid =
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
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 2000 rpm in a swinging bucket centrifuge.  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. Replace cells in 28° shaker until the complexes are ready for transfection
5. Dilute ~10 μg 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 =
2. Determine the volume of cells required for the day
Viability of cells =
: Each transfection requires 15 mL of SF9 cells at 1*10^6 cells/mL, or 1.5*10^7 cells
Cell Density = (mean # of cells per grid)*5*10^4 cells/mL =
 
9. Dispense 12 mL of cell culture into a plastic 125 mL culture flask (disposable flask with a vented top; 0.2 μm pore)
3. Wash the cells by spinning 10 min at 500 rpm in a 15 mL conical in a Beckman TJ-6 centrifuge 
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.
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
 
8. 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.
 
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

Latest revision as of 11:38, 19 February 2013


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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

8. 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.

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

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