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

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…

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