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

Inoculate the bacterial strain one day before the infection in 3 milliliters of Luria-Bertani medium supplemented with streptomycin and ampicillin in a shaker at 37 degrees Celsius overnight. To account for the individual conditions, add coverslips to the wells of a 24-well plate that will be used as controls.

To plate the cells for infection, feed wild-type bone marrow-derived macrophages or BMDMs at a density of 0.125 million cells per well in 1 milliliter of primary macrophage medium. Then, incubate the cells overnight in an incubator at 37 degrees Celsius and 5% carbon dioxide.

The next day, determine the concentration of Salmonella in the overnight culture by measuring the optical density at 600 nanometers or OD₆₀₀ of the diluted culture against phosphate-buffered saline. This ensures that the OD₆₀₀ is measured in the linear range of the spectrophotometer.

Calculate the concentration of Salmonella per milliliter. An OD₆₀₀ of one corresponds to 1 billion bacteria. Dilute the bacteria in macrophage medium to reach a Salmonella concentration of 1.25 million cells per milliliter.

To infect the cells with Salmonella, remove the medium from the BMDMs. Add 1 milliliter of macrophage medium to the uninfected control wells B1 through B3 and C1 through C3, then, add 1 milliliter of Salmonella suspension to wells A1 through A5 to reach a multiplicity of infection of 10 bacteria per cell.

Centrifuge the plate for 15 minutes at 300 times g and 37 degrees Celsius to synchronize the infection before transferring the plate to incubate at 37 degrees Celsius and 5% carbon dioxide. At one hour post-infection, remove the plate from the incubator to add 0.1 milliliters of macrophage medium containing gentamicin to kill extracellular bacteria.

Add gentamicin to all the wells, even the uninfected controls, to ensure that all cells are treated the same way. Then, transfer the plate to an incubator at 37 degrees Celsius and 5% carbon dioxide. At two hours post-infection, remove the plate from the incubator, and wash all wells two times with 1 milliliter of pre-warmed Dulbecco's Modified Eagle's Medium or DMEM.

Replace the DMEM with pre-warmed macrophage medium containing gentamicin to prevent growth of any remaining extracellular bacteria before transferring the plate back to the incubator. Just before the desired time point of analysis, prepare fresh solutions of KHM buffer, KHM buffer with digitonin, and KHM buffer with saponin, in addition to antibody solutions, as described in the text protocol.

To perform cell permeabilization, remove the plate from the incubator and wash the wells three times with 0.5 milliliters of pre-warmed KHM buffer. Then, remove the KHM buffer and add 0.25 milliliters of plain KHM buffer to wells A4, B1, and C1, KHM buffer with digitonin to wells A1 through A3, B2, and C2, and KHM buffer with saponin to wells A5, B3, and C3.

Incubate for exactly 1 minute at room temperature before immediately washing all wells three times with 0.5 milliliters of pre-warmed KHM buffer. Finally, remove the KHM buffer, and add the primary antibody solutions to the appropriate wells before incubating the plate for 15 minutes at 37 degrees Celsius and 5% carbon dioxide.

Following incubation, wash the cells once with 1 milliliter of PBS. For the infected cells in wells A1 through A5, prepare for FACS analysis by washing the cells three times with PBS. Then, add 0.5 milliliter of ice-cold PBS, containing 0.1% Triton.

Next, transfer the cell suspension into a 5-milliliter FACS tube with a cell strainer cap to break up cell aggregates, while keeping samples on ice. Analyze the samples on a FACS machine. Using the fully permeabilized controls, set the gate for total bacteria based on forward and side scatter and the mCherry signal.

Next, analyze the FITC signal in the total bacterial population using the fully-permeabilized and not-permeabilized controls to set the gates for cytosolic bacteria and vacuolar bacteria. With the gates set, analyze the samples and determine the percentage of cytosolic versus vacuolar bacteria using FlowJo software according to the manufacturer's protocol.