2. Detach cells from plates by adding 0.5 mL of 0.05% trypsin per
well and incubate in a 5% CO 2 incubator at 37
C for 3–5 min
(see Note 10).
3. Add 0.5 mL of complete cell culture media to each well to
deactivate trypsin and transfer the contents of each well into a
15 mL Falcon tube.
4. Centrifuge the 15 mL tubes at 225 Â g for 10 min at room
temperature.
5. Gently aspirate the supernatant using a serological pipette
ensuring that the cell pellet is not disturbed.
6. Resuspend the cell pellet by transferring 1 mL of 1Â PBS into
the 15 mL Falcon tube using a micropipette. Repeatedly
pipette gently to ensure thorough resuspension (see Note 21).
7. Store the resuspended cells on ice away from light until they are
analyzed using a flow cytometer (see Note 22).
8. Analyze cells for fluorescence resulting from successful reporter
gene expression (see Note 23).
4 Notes
1. Reconstituted BHI growth media should always be sterilized.
Media can be prepared and stored for future experiments but is
recommended to be made fresh.
2. Plasmid pEGFPN2 can be substituted with any preferred
eukaryotic expression vector or reporter construct.
3. Other cell lines that express β1 integrins on the cell membrane
may be permissive for use with this invasive E. coli vector.
4. The base growth media used for each experiment is cell line
specific. Most immortalized cell lines are maintained in RPMI
or DMEM. It is important that media used for invasion does
not contain serum as this leads to clumping of the bacterial
vectors, which drastically reduces their invasion capabilities.
5. Complete cell culture media used to promote recovery of cells
following the invasion process should contain all necessary
components including 2 mM L-glutamine and 10% FBS. It is
helpful to increase the serum concentration to 12% following
the invasion process to improve recovery rates and limit cell
death.
6. A sterile pipette tip or inoculation loop should be used to
transfer colonies from the plate of BHI agar into liquid BHI
broth. Aeration can be provided by leaving the caps of the
50 mL centrifuge tube slightly open. Culture tubes should be
slightly slanted during incubation.
Enhancing Bactofection with Exogenous Reagents
23
well and incubate in a 5% CO 2 incubator at 37
C for 3–5 min
(see Note 10).
3. Add 0.5 mL of complete cell culture media to each well to
deactivate trypsin and transfer the contents of each well into a
15 mL Falcon tube.
4. Centrifuge the 15 mL tubes at 225 Â g for 10 min at room
temperature.
5. Gently aspirate the supernatant using a serological pipette
ensuring that the cell pellet is not disturbed.
6. Resuspend the cell pellet by transferring 1 mL of 1Â PBS into
the 15 mL Falcon tube using a micropipette. Repeatedly
pipette gently to ensure thorough resuspension (see Note 21).
7. Store the resuspended cells on ice away from light until they are
analyzed using a flow cytometer (see Note 22).
8. Analyze cells for fluorescence resulting from successful reporter
gene expression (see Note 23).
4 Notes
1. Reconstituted BHI growth media should always be sterilized.
Media can be prepared and stored for future experiments but is
recommended to be made fresh.
2. Plasmid pEGFPN2 can be substituted with any preferred
eukaryotic expression vector or reporter construct.
3. Other cell lines that express β1 integrins on the cell membrane
may be permissive for use with this invasive E. coli vector.
4. The base growth media used for each experiment is cell line
specific. Most immortalized cell lines are maintained in RPMI
or DMEM. It is important that media used for invasion does
not contain serum as this leads to clumping of the bacterial
vectors, which drastically reduces their invasion capabilities.
5. Complete cell culture media used to promote recovery of cells
following the invasion process should contain all necessary
components including 2 mM L-glutamine and 10% FBS. It is
helpful to increase the serum concentration to 12% following
the invasion process to improve recovery rates and limit cell
death.
6. A sterile pipette tip or inoculation loop should be used to
transfer colonies from the plate of BHI agar into liquid BHI
broth. Aeration can be provided by leaving the caps of the
50 mL centrifuge tube slightly open. Culture tubes should be
slightly slanted during incubation.
Enhancing Bactofection with Exogenous Reagents
23