3.6 Gene Delivery
Assessment
1. Incubate RAW264.7 macrophage cells with murine microphage medium (Subheading 2.3) in T75 flasks and culture at
37
C in a humidified incubator at a 5% CO 2 level until cells
become confluent, passaging the medium and cells as needed.
2. Remove the medium via aspiration with a pipette and wash the
cells with 1Â volume of PBS buffer (pH 7.4) one time.
3. Detach the cells from the surface by incubating the cells with
Express enzyme solution for 10 min.
4. To quench the trypsin reaction, add 10 mL of complete cell
culture and mix by pipetting.
5. Measure the cell density using a hemocytometer (see Note 10).
6. Transfer the cells to a 15 mL conical tube and centrifuge at
500 Â g for 5 min at 18
C.
7. Remove the supernatant and then resuspend the pellet in
antibiotic-free medium to reach 3 Â 10
4 cells per 100 μL.
8. Transfer 100 μL of the cell solution into each well of two
different types of 96-well plates (a clear, culture-treated format
and a flat-bottomed, sterile, white polystyrene format) and
incubate for 24 h at 30
C.
9. Culture bacterial strains to be used for gene delivery as
described for the DNA and protein release assay (10 mL
media) and induce for an hour with 100 μM of IPTG at 22
C.
10. Harvest bacterial cells via centrifugation at 4
C and
13,000 rpm in a microcentrifuge for 10 min.
11. Wash the bacterial pellet washed using 10 mL of PBS.
12. Following the wash with PBS, standardize the cell density to an
OD 600nm of 0.5 in PBS.
13. Dilute the bacterial cells in antibiotic-free RPMI-1600 in different ratios to create multiple bacteria-to-macrophage multiplicity of infection (MOI) ratios (1:1, 10:1, 100:1, 250:1,
500:1, 750:1, 1000:1, 1250:1, 1500:1, and 2000:1) (see
Note 11).
14. Afterwards, remove the macrophage medium in both 96-well
plates and replaced it with 50 μL of each respective bacterialRPMI dilution (to generate the resulting MOI values) and
incubate for an hour at 30
C in a 5% CO 2 incubator. Also
include a sample of macrophage which have no bacteria added
as a negative control (see Note 12).
15. Add 50 μL of gentamicin-containing RPMI 1640 (100 mg/L)
to each well and incubate for 24 h at 30
C.
16. Quantify the luciferase expression using the Bright Glo assay
kit (Promega) following the manufacturer’s instructions.
17. Calculate gene delivery by dividing luciferase expression by
protein content for each well/plate (Fig. 5).
Bactofection Enhancement with ΦX174 Gene E
11
Assessment
1. Incubate RAW264.7 macrophage cells with murine microphage medium (Subheading 2.3) in T75 flasks and culture at
37
C in a humidified incubator at a 5% CO 2 level until cells
become confluent, passaging the medium and cells as needed.
2. Remove the medium via aspiration with a pipette and wash the
cells with 1Â volume of PBS buffer (pH 7.4) one time.
3. Detach the cells from the surface by incubating the cells with
Express enzyme solution for 10 min.
4. To quench the trypsin reaction, add 10 mL of complete cell
culture and mix by pipetting.
5. Measure the cell density using a hemocytometer (see Note 10).
6. Transfer the cells to a 15 mL conical tube and centrifuge at
500 Â g for 5 min at 18
C.
7. Remove the supernatant and then resuspend the pellet in
antibiotic-free medium to reach 3 Â 10
4 cells per 100 μL.
8. Transfer 100 μL of the cell solution into each well of two
different types of 96-well plates (a clear, culture-treated format
and a flat-bottomed, sterile, white polystyrene format) and
incubate for 24 h at 30
C.
9. Culture bacterial strains to be used for gene delivery as
described for the DNA and protein release assay (10 mL
media) and induce for an hour with 100 μM of IPTG at 22
C.
10. Harvest bacterial cells via centrifugation at 4
C and
13,000 rpm in a microcentrifuge for 10 min.
11. Wash the bacterial pellet washed using 10 mL of PBS.
12. Following the wash with PBS, standardize the cell density to an
OD 600nm of 0.5 in PBS.
13. Dilute the bacterial cells in antibiotic-free RPMI-1600 in different ratios to create multiple bacteria-to-macrophage multiplicity of infection (MOI) ratios (1:1, 10:1, 100:1, 250:1,
500:1, 750:1, 1000:1, 1250:1, 1500:1, and 2000:1) (see
Note 11).
14. Afterwards, remove the macrophage medium in both 96-well
plates and replaced it with 50 μL of each respective bacterialRPMI dilution (to generate the resulting MOI values) and
incubate for an hour at 30
C in a 5% CO 2 incubator. Also
include a sample of macrophage which have no bacteria added
as a negative control (see Note 12).
15. Add 50 μL of gentamicin-containing RPMI 1640 (100 mg/L)
to each well and incubate for 24 h at 30
C.
16. Quantify the luciferase expression using the Bright Glo assay
kit (Promega) following the manufacturer’s instructions.
17. Calculate gene delivery by dividing luciferase expression by
protein content for each well/plate (Fig. 5).
Bactofection Enhancement with ΦX174 Gene E
11