Using an automated cell counter, adjust the prepared RAW 264.7 cells to the concentration of 1 x 106 cells per milliliter. Add 1 milliliter of the sample to wells of a 6-well tissue culture plate. Infect the cells with prepared Lm inoculum corresponding to a multiplicity of infection of 50.
At 1.5 hours post-infection, in a 1.5-millimeter microcentrifuge tube filled with 500 microliters of 4% PFA, collect media from the first set of wells, and place the tube on ice. To collect intracellular Lm, add 1 milliliter of distilled sterile water to each well. After 60 seconds, transfer the resulting water lysate to a 1.5-milliliter microcentrifuge tube with 500 microliters of 4% PFA. Vortex the tube vigorously for 10 seconds and place it on ice.
For remaining wells, remove media and replace with 1 milliliter of DMEM/FCS with 5 micrograms per milliliter gentamicin to kill extracellular Lm. Promptly return the dish to the incubator.
After 30 minutes, remove media and replace with DMEM/FCS without gentamicin. After another two and four hours, collect media in lysates for the second and third time points into the tubes and place them on the ice to chill. Centrifuge tubes at 10,000 times g for 7 minutes.
Using a pipette, remove supernatant without disturbing the pellet. Add staining cocktail containing 50 microliters of 4% PFA and 15 microliters of phalloidin into the tube, and mix to resuspend each pellet. Incubate tubes in the dark at 4 degrees Celsius for 20 minutes.
After incubation, add 1 milliliter of FACS buffer to each tube and pipette up and down to mix. Spin tubes in the centrifuge at 10,000 times g for 7 minutes. Remove supernatant without disturbing the pellet.
For immediate use, suspend each pellet in 400 microliters of FACS buffer. If storing for later analysis, add 200 microliters of FACS buffer, and 200 microliters of 4% PFA into the tube and mix to suspend.
