2. Transfer 10 μL bacteria that has been harvested during the late
log growth phase and resuspended in invasion media into
30 μL of invasion media containing Lipofectamine 2000 or
PULSin and vortex lightly or mix using a pipette. Incubate
mixture at room temperature for 15 min before invading of
host cells (see Note 15).
3. To include antimicrobial reagents to the invasive E. coli–lipid
complex (see Fig. 1), dilute antibiotics including amantadine,
chloroquine, tetracycline, and polymyxin B to their working
concentration (see Subheading 2.2) in 950 μL of invasion
media in a 1.5 mL centrifuge tube. Vortex thoroughly to mix.
Incubate at room temperature for 10 min.
4. Transfer 40 μL of E. coli–lipid complexes (from Subheading
3.3, step 1) or 10 μL of harvested invasive E. coli (from Subheading 3.1, step 5) into the 1.5 mL centrifuge tube containing diluted antibiotics immediately prior to invasion of host
cells. Mix thoroughly by pipetting.
3.4 E. coli Invasion
and Co-delivery with
transfection Reagents
1. Top up the mixture containing invasive E. coli–lipid complexes
or E. coli–lipid–antibiotic solution to 1 mL using invasion
media and mix by pipetting (see Note 16).
2. Wash cells twice by gently pipetting 3 mL of 1Â PBS into
6-well plates and gently swirling to mix. Remove PBS using a
serological pipette (see Note 17).
3. Transfer 1 mL of E. coli–lipid complexes from the 1.5 mL
microcentrifuge tube onto the monolayer of cells in each
well. Swirl plates gently to mix.
4. Centrifuge 6-well plates in a plate centrifuge at 225 Â g for
10 min. Immediately transfer plates to a 5% CO 2 incubator set
at 37
C with humidity and incubate for 1 h (see Note 18).
5. Aspirate media from wells using a serological pipette. Gently
rinse each well with 3 mL of 1Â PBS (see Note 19).
6. Add 1 mL of complete cell culture media containing 80 μg/mL
of gentamicin to each well. Incubate for 1 h at 37
C in a CO 2
incubator with humidity.
7. After incubation, repeat step 6 three times to remove
gentamicin.
8. Add 1 mL of complete cell culture media containing 20 μg/mL
of gentamicin. Incubate at 37
C in a CO 2 incubator with
humidity for 24–48 h to analyze gene expression (see Note 20).
3.5 Detecting Gene
Expression
1. Remove complete culture media containing 20 μg/mL gentamicin using a serological pipette. Rinse cells 2–3 times using
1Â PBS that has been prewarmed to 37
C using a water bath.
22
Andrew N. Osahor and Kumaran Narayanan
log growth phase and resuspended in invasion media into
30 μL of invasion media containing Lipofectamine 2000 or
PULSin and vortex lightly or mix using a pipette. Incubate
mixture at room temperature for 15 min before invading of
host cells (see Note 15).
3. To include antimicrobial reagents to the invasive E. coli–lipid
complex (see Fig. 1), dilute antibiotics including amantadine,
chloroquine, tetracycline, and polymyxin B to their working
concentration (see Subheading 2.2) in 950 μL of invasion
media in a 1.5 mL centrifuge tube. Vortex thoroughly to mix.
Incubate at room temperature for 10 min.
4. Transfer 40 μL of E. coli–lipid complexes (from Subheading
3.3, step 1) or 10 μL of harvested invasive E. coli (from Subheading 3.1, step 5) into the 1.5 mL centrifuge tube containing diluted antibiotics immediately prior to invasion of host
cells. Mix thoroughly by pipetting.
3.4 E. coli Invasion
and Co-delivery with
transfection Reagents
1. Top up the mixture containing invasive E. coli–lipid complexes
or E. coli–lipid–antibiotic solution to 1 mL using invasion
media and mix by pipetting (see Note 16).
2. Wash cells twice by gently pipetting 3 mL of 1Â PBS into
6-well plates and gently swirling to mix. Remove PBS using a
serological pipette (see Note 17).
3. Transfer 1 mL of E. coli–lipid complexes from the 1.5 mL
microcentrifuge tube onto the monolayer of cells in each
well. Swirl plates gently to mix.
4. Centrifuge 6-well plates in a plate centrifuge at 225 Â g for
10 min. Immediately transfer plates to a 5% CO 2 incubator set
at 37
C with humidity and incubate for 1 h (see Note 18).
5. Aspirate media from wells using a serological pipette. Gently
rinse each well with 3 mL of 1Â PBS (see Note 19).
6. Add 1 mL of complete cell culture media containing 80 μg/mL
of gentamicin to each well. Incubate for 1 h at 37
C in a CO 2
incubator with humidity.
7. After incubation, repeat step 6 three times to remove
gentamicin.
8. Add 1 mL of complete cell culture media containing 20 μg/mL
of gentamicin. Incubate at 37
C in a CO 2 incubator with
humidity for 24–48 h to analyze gene expression (see Note 20).
3.5 Detecting Gene
Expression
1. Remove complete culture media containing 20 μg/mL gentamicin using a serological pipette. Rinse cells 2–3 times using
1Â PBS that has been prewarmed to 37
C using a water bath.
22
Andrew N. Osahor and Kumaran Narayanan