2.3.3 Cell Culture
Materials
1. Six-well cell culture plates.
2. T-75 Cell culture flask.
3. 5 mL serological pipettes.
4. 10 mL serological pipettes.
5. Automatic pipettor.
6. Plate centrifuge.
7. Trypan blue.
8. Hemocytometer.
9. 15 mL Falcon centrifuge tubes.
10. 50 mL Falcon centrifuge tubes.
11. Water bath.
3 Methods
All procedures should be carried out at room temperature unless
indicated otherwise.
3.1 Invasive E. coli
Growth
1. Spread E. coli on freshly prepared BHI agar and incubate at
37
C overnight until single colonies are obtained.
2. Inoculate 20 mL of sterile BHI broth with freshly grown single
colonies of invasive E. coli DH10B (pGB2Ωinv-hly,
pEGFPN2) and/or control noninvasive E. coli DH10B
(pEGFPN2) (see Note 6).
3. Grow 10 mL of E. coli cultures overnight in 5 mL of BHI broth
in a sterile 50 mL Falcon tube at 37
C with shaking at 220 rpm
overnight, or until late log/stationary phase is achieved
approximately 18 h post-inoculation or until OD 600 of 2.4
(see Note 7).
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 (see Note 8).
5. Resuspend the bacterial pellet in 0.9 mL of invasion media and
vortex thoroughly 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 (see Note 9).
2. Using a 5 mL serological pipette, gently remove cells from the
cryopreservation vial into a T-75 cell culture flask containing
complete cell culture media prewarmed to 37
C in a 5% CO 2
incubator.
20
Andrew N. Osahor and Kumaran Narayanan
Materials
1. Six-well cell culture plates.
2. T-75 Cell culture flask.
3. 5 mL serological pipettes.
4. 10 mL serological pipettes.
5. Automatic pipettor.
6. Plate centrifuge.
7. Trypan blue.
8. Hemocytometer.
9. 15 mL Falcon centrifuge tubes.
10. 50 mL Falcon centrifuge tubes.
11. Water bath.
3 Methods
All procedures should be carried out at room temperature unless
indicated otherwise.
3.1 Invasive E. coli
Growth
1. Spread E. coli on freshly prepared BHI agar and incubate at
37
C overnight until single colonies are obtained.
2. Inoculate 20 mL of sterile BHI broth with freshly grown single
colonies of invasive E. coli DH10B (pGB2Ωinv-hly,
pEGFPN2) and/or control noninvasive E. coli DH10B
(pEGFPN2) (see Note 6).
3. Grow 10 mL of E. coli cultures overnight in 5 mL of BHI broth
in a sterile 50 mL Falcon tube at 37
C with shaking at 220 rpm
overnight, or until late log/stationary phase is achieved
approximately 18 h post-inoculation or until OD 600 of 2.4
(see Note 7).
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 (see Note 8).
5. Resuspend the bacterial pellet in 0.9 mL of invasion media and
vortex thoroughly 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 (see Note 9).
2. Using a 5 mL serological pipette, gently remove cells from the
cryopreservation vial into a T-75 cell culture flask containing
complete cell culture media prewarmed to 37
C in a 5% CO 2
incubator.
20
Andrew N. Osahor and Kumaran Narayanan