3. Incubate cells in a 37
C incubator with 5% CO 2 with humidity
overnight.
4. Wash cells with 1Â PBS that has been prewarmed to 37
C by
gently pipetting onto the walls of the culture vessel and gently
swirling to ensure proper washing. Transfer 15 mL of prewarmed complete culture media to the culture vessel. Grow
cells until 70% confluent.
5. Aspirate spent media from the culture vessel using a serological
pipette. Wash cells twice using 1Â PBS by gently pipetting onto
the walls of the culture vessel and gently swirling to ensure
proper washing.
6. Remove 1Â PBS using a serological pipette and transfer 4 mL
of 0.05% trypsin to detach cells from the culture flask. Incubate
cells in trypsin solution at 37
C with 5% CO 2 and humidity for
approximately 3–5 min or until cells are completely detached
(see Note 10).
7. Gently add 5 mL of complete media to the culture vessel using
a serological pipette to deactivate trypsin (see Note 11).
8. Collect detached cells into a 15 mL Falcon tube using a serological pipette. Centrifuge the cells at 225 Â g for 5 min at
room temperature.
9. Remove the supernatant using a serological pipette (see Note
12).
10. Resuspend the cell pellet in 1 mL of complete cell culture
media. Pipette gently to ensure homogenous resuspension.
Do not vortex.
11. Remove 10 μL of cells onto a hemocytometer using a micropipette and mix with 10 μL trypan blue dye to obtain a viable cell
count.
12. Prepare cells for invasion by seeding HEK 293, HeLa, or
HT1080 cells in 6-well plates at a density of 1 Â 10
5 cells per
well 24 h before invasion experiments (see Note 13).
13. On the day of invasion, remove growth media from the cell
culture vessel using a sterile serological pipette and rinse cells
twice with 1Â PBS that has been prewarmed to 37
C using a
sterile water bath.
14. Remove 1Â PBS from culture vessel using a serological pipette
immediately before invasion (see Note 14).
3.3 Complexing E.
coli with Invasion
Enhancing Reagents
1. Dilute Lipofectamine 2000 and/or PULSin in invasion media
that does not contain serum (see Note 4). Add 1 μL of either
lipid to 30 μL of invasion media in a 1.5 mL centrifuge tube.
Vortex thoroughly to ensure mixing. Incubate at room temperature for 10 min before complexation with invasive vector.
Enhancing Bactofection with Exogenous Reagents
21
C incubator with 5% CO 2 with humidity
overnight.
4. Wash cells with 1Â PBS that has been prewarmed to 37
C by
gently pipetting onto the walls of the culture vessel and gently
swirling to ensure proper washing. Transfer 15 mL of prewarmed complete culture media to the culture vessel. Grow
cells until 70% confluent.
5. Aspirate spent media from the culture vessel using a serological
pipette. Wash cells twice using 1Â PBS by gently pipetting onto
the walls of the culture vessel and gently swirling to ensure
proper washing.
6. Remove 1Â PBS using a serological pipette and transfer 4 mL
of 0.05% trypsin to detach cells from the culture flask. Incubate
cells in trypsin solution at 37
C with 5% CO 2 and humidity for
approximately 3–5 min or until cells are completely detached
(see Note 10).
7. Gently add 5 mL of complete media to the culture vessel using
a serological pipette to deactivate trypsin (see Note 11).
8. Collect detached cells into a 15 mL Falcon tube using a serological pipette. Centrifuge the cells at 225 Â g for 5 min at
room temperature.
9. Remove the supernatant using a serological pipette (see Note
12).
10. Resuspend the cell pellet in 1 mL of complete cell culture
media. Pipette gently to ensure homogenous resuspension.
Do not vortex.
11. Remove 10 μL of cells onto a hemocytometer using a micropipette and mix with 10 μL trypan blue dye to obtain a viable cell
count.
12. Prepare cells for invasion by seeding HEK 293, HeLa, or
HT1080 cells in 6-well plates at a density of 1 Â 10
5 cells per
well 24 h before invasion experiments (see Note 13).
13. On the day of invasion, remove growth media from the cell
culture vessel using a sterile serological pipette and rinse cells
twice with 1Â PBS that has been prewarmed to 37
C using a
sterile water bath.
14. Remove 1Â PBS from culture vessel using a serological pipette
immediately before invasion (see Note 14).
3.3 Complexing E.
coli with Invasion
Enhancing Reagents
1. Dilute Lipofectamine 2000 and/or PULSin in invasion media
that does not contain serum (see Note 4). Add 1 μL of either
lipid to 30 μL of invasion media in a 1.5 mL centrifuge tube.
Vortex thoroughly to ensure mixing. Incubate at room temperature for 10 min before complexation with invasive vector.
Enhancing Bactofection with Exogenous Reagents
21