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

Abstract

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)

In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

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....

Materials

List of materials used in this article

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