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

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
BHI (Brain Heart Infusion) broth	EMD Milipore	110493
Cell Strainer, 70 µm	VWR	10199-656
Collagenase D	Roche	11088858001
DMEM media	Gibco	11965-092
FACS tubes	BD Falcon	352054
FBS - Heat-Inactivated	Sigma-Aldrich	F4135-500ML
Hanks' Balanced Salt solution	Sigma-Aldrich	H6648-6X500ML
LB agar	Grow Cells MS	MBPE-4040
LIVE/DEAD Fixable Yellow Dead Cell Stain kit	Life Technologies	L349S9
Rhodamine Phalloidin	Thermo Fischer	R415
SP6800 Spectral Analyzer	Sony
TPP Tissue Culture 48-Well Plates	MIDSCI	TP92048
TPP Tissue Culture 6-Well Plates	MIDSCI	TP92406