Infection of Epithelial Cells With Salmonella enterica - Bacterial Pathogenesis: Methods and Protocols - page 194

3. Methods
3.1. Preparation of HeLa Cells for Infection
1. Grow HeLa cells in a humidified 37 °C, 5% CO 2 tissue-culture incubator. Low
passage number (<15 after receipt from ATCC) cells should be used ( see Note 7 ).
2. Passage cells when they are actively growing ( see Note 8 ).
3. For cells grown in a 75-cm 2 flask, decant media and rinse monolayer with

5 mL PBS.
4. Immediately add 5 mL Trypsin/EDTA and gently swirl over the monolayer.
Remove 4 mL of the Trypsin/EDTA and leave cells at room temperature for up
to 5 min. The cells should be easily detached by tapping the flask firmly, but
gently, against an open hand.
5. Immediately add 5 mL of GM and resuspend the cells by pipetting up and down
with a 5-mL pipette.
6. Count the cells using a hemocytometer. For infection experiments, seed in 24-well
(5×10 4 per well) or 6-well dishes (2 × 10 5 per well) and grow overnight (14-20
h) before infection.
7. Cells should still be actively growing, in logarithmic phase, at the time of infection.

3.2. Preparation of SPI1-Induced Salmonella
1. Grow S. enterica serovar Typhimurium overnight (16-18 h) in 1.5-2 mL LB in a
15-mL tube with loose cap. Incubate at 37 °C in a shaking incubator (225 rpm).
2. Subculture Salmonella by transferring 300 μL of the overnight culture into 10 mL
of LB in a loosely capped 125-mL Erlenmeyer flask. Incubate at 37 °C in a
shaking incubator (25 rpm) for 3.5 h or to late log phase ( see Note 9 ). At this
time, there should be approximately3×10 9 CFU/mL ( see Note 10 ).
3. Pellet 1 mL of the Salmonella subculture by centrifugation at 1000 × g in a
microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL HBSS.
5. Use immediately.

3.3. Invasion
1. Inoculate cells with Salmonella (MOI 10-100) by adding bacteria directly to the
cell-culture supernatant ( see Note 11 ).
2. Incubate for 2-10 min at 37 °C in 5% CO 2 .
3. Aspirate media and rinse the monolayer twice with PBS, or HBSS, to remove
extracellular bacteria.
4. Add fresh GM and incubate for 20 min at 37 °C in 5% CO 2 .
5. Replace GM with fresh GM containing 50 μg/mL gentamicin.
6. Incubate for 40 min at 37 °C in 5% CO 2 .
7. Replace GM with fresh GM containing 5 μg/mL gentamicin for remainder of
experiment.