Infection of Epithelial Cells With Salmonella enterica - Bacterial Pathogenesis: Methods and Protocols - page 195

3.4. Gentamicin Protection Assay for Quantification
of Intracellular Bacteria
1. Aspirate media and rinse the monolayer once with PBS to remove gentamicin.
2. Solubilize the monolayer in 1 mL solubilization buffer.
3. Immediately transfer tomicrofuge tubes and dilute in PBS (1:10, 1:100, and 1:1000).
4. Spot 10 μL of each dilution on dry LB plates ( see Fig. 2 ).
5. Incubate overnight at 37 °C.
6. Count colonies as follows:

a. if three colonies in 10 μL of undiluted, then total cfu/well=3×100=300.

b. if three colonies in 10 μL of 1:1000, then total cfu/well=3×100×1000 =

3×10 5 .

3.5. Immunofluorescence “Inside-Outside” Assay
1. Passage cells as described above, except grow in 24-well plates with a glass
coverslip in the bottom of each well. Make sure that the coverslips are not
floating by gently pushing each one to the bottom with a pipette tip.
2. Infect cells 16-20 h after passaging as above.
3. Rinse monolayer once with HBBS.
4. Fix in fresh 2.5% formaldehyde (warmed to 37 °C), 300-500 μL/well, for 10 min
at room temperature. Discard the formaldehyde appropriately.
5. Rinse twice with PBS (use “squirt bottle” and direct jet onto side of well).
6. Wash three times for 10 min each with PBS.
7. Remove PBS thoroughly by aspiration and make sure coverslip is not touching
sides of well.

Fig. 2. Schematic diagram of an LB plate used to estimate colony-forming units in
a cell lysate. See text for explanation.