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).
1. Digitonin Assay with mCherry-expressing Salmonella (FACS-based Analysis)
Table 1: Plating scheme for bone-marrow-derived macrophages (BMDMs) in a 24-well format for subsequent infection with Salmonella, permeabilization, and staining.
Permeabilization | Staining | ||||||
Glass coverslip | Salmonella | Digitonin | Saponin | anti-Salmonella | anti-Calnexin | anti-PDI | |
A1 (sample) | -/+* | + | + | + | |||
A2 (sample) | -/+* | + | + | + | |||
A3 (sample) | -/+* | + | + | + | |||
A4 (no permeabilization control) | -/+* | + | + | ||||
A5 (complete permeabilization control) | -/+* | + | + | + | |||
B1 (stained for calnexin) | + | + | |||||
B2 (stained for calnexin) | + | + | + | ||||
B3 (stained for calnexin) | + | + | + | ||||
C1 (stained for PDI) | + | + | |||||
C2 (stained for PDI) | + | + | + | ||||
C3 (stained for PDI) | + | + | + |
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 permeabilized, while positive control cells are completely permeabilized before antibody staining.
Figure 2. FACS analysis of fully permeabilized, unpermeabilized, or differentially permeabilized samples infected for 6 hr with mCherry+ wild-type or ΔsifA Salmonella for 6 hr.
The authors have nothing to disclose.
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 |