Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

1. Sampling Processing and Analysis of Intracellular and Emergent Lm by Flow Cytometry (FCM)

1. Prepare mouse macrophage-like cell line RAW 264.7 in DMEM tissue culture media supplemented with 10% fetal calf serum (henceforth referred to as DMEM/FCS) using a 125 mL flask and a tissue culture incubator maintained at 37 °C/5% CO₂. Adjust cells to 1 x 10⁶ cells/mL and add 1 mL (1 x 10⁶ cells) to wells of a 6-well tissue culture dish.
2. Prepare Listeria monocytogenes, Lm, bacterium inoculum and infect cells with the volume of inoculum corresponding to an MOI of 50 (5 x 10⁷ CFU).
3. At 1.5 hours post-infection (hpi), collect media from the first set of wells and place in a 1.5 mL microcentrifuge tube with 500 µL of 4% paraformaldehyde (PFA) and place on ice until all wells have been processed.
4. To collect intracellular Lm, add 1 mL of ds H₂O to the well and after 60 s, transfer the resulting water lysate to a 1.5 mL microcentrifuge tube with 500 µL of 4% PFA. Vortex vigorously for 10 seconds and place on ice until all wells have been processed.
5. For remaining wells, remove media and replace with 1 mL of DMEM/FCS with 5 µg/mL gentamicin to kill extracellular Lm and promptly return cells to the incubator.
6. After 30 min (2 hpi) remove media and replace with DMEM/FCS without gentamicin.
7. After 2 and 4 h (4 and 6 hpi) take second and third time points by collecting media and lysates.
8. Centrifuge tubes at 10,000 x g for 7 min. Remove supernatant without disturbing the pellet.
9. Suspend each pellet in a staining cocktail of 50 µL of 4% PFA and 15 µL of phalloidin. Incubate tubes in the dark at 4 °C for 20 min.
10. After incubation add 1 mL of FACS buffer to each tube and pipette up and down to mix.
11. Spin tubes at 10,000 x g for 7 min. Remove supernatant without disturbing the pellet.
12. Suspend each pellet in 400 µL of FACS buffer for immediate use, or 200 µL of FACS buffer and 200 µL of 4% PFA if storing for later analysis.
13. Run samples on flow cytometry and analyze the collected data.