An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

Published: February 29, 2024

Abstract

Source: Meunier, E., et al. Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization. J. Vis. Exp. (2015).

This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin, leaving the SCV intact. Upon labeling the cytosolic bacteria with a fluorescently tagged antibody, the percentage of vacuolar and cytosolic bacteria is quantified using fluorescence-activated cell sorting (FACS).

Protocol

1. Digitonin Assay with mCherry-expressing Salmonella (FACS-based Analysis) Preparing bacteria Note: Salmonella wild-type SL1344 (Streptomycin resistant) expressing mCherry from a plasmid (pFPV encoding mCherry under the rpsM promoter) is used in this assay. Bacterial phagocytosis and proliferation were not altered by the constitutive expression of mCherry (data not shown). Inoculate the strain 1 day before the infection in 3 ml of LB medi…

Representative Results

Figure 1. Schematic representation of protocol 1. Infected cells containing mCherry-positive Salmonella either in intact vacuoles or the cytosol are differentially permeabilized with digitonin. Cytosolic Salmonella are stained with anti-Salmonella coupled to FITC. Cells are washed and lysed with Triton X-100 for FACS analysis. Negative control cells are not permeabilize…

Offenlegungen

The authors have nothing to disclose.

Materials

Digitonin Sigma D5628 50ug/mL
PFA mpbio 219998380
HEPES Life Technologies 15630
Potassium Acetate Sigma 791733
MgCl2 Sigma M8266
BSA Sigma A2153
anti-Salmonella CSA-1-FITC KPL 01-91-99-MG 1/500
anti-Salmonella CSA-1 KPL 02-91-99-MG 1/500
anti-Calnexin Enzo Lifesciences SPA-860D 1/100
anti-PDI Enzo Lifesciences SPA-890 1/100
Saponin Sigma 47036
Vectashield mounting medium Vectorlabs H-1200
Anti Rabbit antibody-488 Molecular Probes A-11070 1/500
Gentamicin Life Technologies 15710-49 100ug/mL and 10ug/mL
Triton X-100 Promega H5141
PBS Gibco 20012-019
DMEM Sigma D6429
NEAA Amimed 5-13K00-H
FACS Fortessa BD technologies detection mCherry 610 nm
FACS Fortessa BD technologies detection FITC 530 nm

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An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages. J. Vis. Exp. (Pending Publication), e21994, doi: (2024).

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