3 Methods
3.1 Invasive E. coli
Preparation
1. Spread E. coli on freshly prepared BHI agar and incubate at
37
C overnight until single colonies are obtained.
2. Inoculate 10 mL of sterile BHI broth with freshly grown single
colonies of invasive E. coli DH10B (pGB2Ωinv-hly,
pUltraRFP-KM) and/or control noninvasive E. coli DH10B
(pEGFPN2).
3. Grow E. coli cultures overnight in 10 mL of BHI broth in a
sterile 50 mL Falcon tube at 37
C with shaking at 220 rpm
until late log/stationary phase is achieved approximately 18 h
post inoculation or until OD 600 reaches approximately 1.4.
4. Harvest bacteria by centrifugation at 6000 Â g for 7 min at
room temperature. Discard the spent bacterial media by gently
pouring out the supernatant.
5. Resuspend the bacterial pellet in 0.9 mL of invasion media and
mix thoroughly by pipetting to ensure the bacterial pellet is
fully dissociated and evenly mixed.
3.2 Mammalian Cell
Growth
1. Transfer a vial of mammalian cells from cryopreservation and
thaw rapidly in a 37
C water bath.
2. Using a 5 mL serological pipette, gently remove the thawed
cells from the cryopreservation vial into a 15 mL Falcon tube
containing 10 mL complete media and pipette gently to mix
the cells. Centrifuge briefly at 200 Â g for 5 min, remove the
supernatant, and resuspend the pellet gently in 5 mL complete
media before transferring to a T-75 cell culture flask containing
15 mL prewarmed complete cell culture media.
3. Incubate the cells in a 37
C incubator with 5% CO 2 with
humidity overnight.
4. Aspirate spent media from the culture vessel using a serological
pipette. Wash cells twice using prewarmed 1Â PBS by gently
pipetting onto the walls of the culture vessel and gently swirling
to ensure proper washing.
5. Remove 1Â PBS using a serological pipette and add 15 mL of
complete culture media to the culture flask. Incubate cells
overnight at 37
C with 5% CO 2 or grow cells in complete
culture media until they reach approximately 70% confluent.
6. Aspirate spent media from the culture vessel using a serological
pipette and wash cells twice using 1Â PBS.
7. Aspirate 1Â PBS from the culture flask and transfer 4 mL of
prewarmed 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.
34
Andrew N. Osahor et al.
3.1 Invasive E. coli
Preparation
1. Spread E. coli on freshly prepared BHI agar and incubate at
37
C overnight until single colonies are obtained.
2. Inoculate 10 mL of sterile BHI broth with freshly grown single
colonies of invasive E. coli DH10B (pGB2Ωinv-hly,
pUltraRFP-KM) and/or control noninvasive E. coli DH10B
(pEGFPN2).
3. Grow E. coli cultures overnight in 10 mL of BHI broth in a
sterile 50 mL Falcon tube at 37
C with shaking at 220 rpm
until late log/stationary phase is achieved approximately 18 h
post inoculation or until OD 600 reaches approximately 1.4.
4. Harvest bacteria by centrifugation at 6000 Â g for 7 min at
room temperature. Discard the spent bacterial media by gently
pouring out the supernatant.
5. Resuspend the bacterial pellet in 0.9 mL of invasion media and
mix thoroughly by pipetting to ensure the bacterial pellet is
fully dissociated and evenly mixed.
3.2 Mammalian Cell
Growth
1. Transfer a vial of mammalian cells from cryopreservation and
thaw rapidly in a 37
C water bath.
2. Using a 5 mL serological pipette, gently remove the thawed
cells from the cryopreservation vial into a 15 mL Falcon tube
containing 10 mL complete media and pipette gently to mix
the cells. Centrifuge briefly at 200 Â g for 5 min, remove the
supernatant, and resuspend the pellet gently in 5 mL complete
media before transferring to a T-75 cell culture flask containing
15 mL prewarmed complete cell culture media.
3. Incubate the cells in a 37
C incubator with 5% CO 2 with
humidity overnight.
4. Aspirate spent media from the culture vessel using a serological
pipette. Wash cells twice using prewarmed 1Â PBS by gently
pipetting onto the walls of the culture vessel and gently swirling
to ensure proper washing.
5. Remove 1Â PBS using a serological pipette and add 15 mL of
complete culture media to the culture flask. Incubate cells
overnight at 37
C with 5% CO 2 or grow cells in complete
culture media until they reach approximately 70% confluent.
6. Aspirate spent media from the culture vessel using a serological
pipette and wash cells twice using 1Â PBS.
7. Aspirate 1Â PBS from the culture flask and transfer 4 mL of
prewarmed 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.
34
Andrew N. Osahor et al.