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

Published: September 29, 2023

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) 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. Prepare …

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro. J. Vis. Exp. (Pending Publication), e21648, doi: (2023).

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