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