After preparing a Vero cell suspension of 105 cells per milliliter in RPMI medium according to the text protocol, dispense 100 microliters of the suspension in each well of a black 96-well plate with a flat transparent bottom. Centrifuge the plate at 400 times g and room temperature for five minutes, and incubate at 37 degrees Celsius and 5% carbon dioxide overnight.
The following day, thaw at room temperature 96-well plates containing the Coxiella mutants and dilute 150 microliters of bacterial suspensions in 300 microliters of RPMI without phenol red and FBS, in a deep-well 96-well plate. Next, remove the medium from the Vero cells and dispense 100 microliters per well of diluted Coxiella mutants. Use the A1 well as a negative control and wells A2 and A3 as positive controls.
Using an aerosol-tight centrifuge plate holder, centrifuge the plate at 400 times g and room temperature for 10 minutes. Then, after incubating the plate at 37 degrees Celsius in a humidified atmosphere of 5% carbon dioxide for two hours, replace the bacteria-containing medium with 100 microliters per well of fresh, complete RPMI medium. With a fluorescence microplate reader and filters for fluorescein, measure GFP fluorescence every day for seven days.
On day seven post-infection, remove the medium from the plate, and replace it with 50 microliters per well of fresh, complete medium containing a 1 to 1000 dilution of cell-permeable fluorescent dye. Incubate the cells for 30 to 60 minutes. After the incubation, replace the medium with 50 microliters per well of 4% PFA in PBS. Incubate at room temperature for 30 minutes, then, remove the PFA-containing buffer, before using PBS to wash the wells three times.
After removing the final wash, dispense 50 microliters of blocking solution to each well and incubate at room temperature for 30 minutes. Then, replace the blocking solution with 40 microliters per well of fresh blocking solution and a 1 to 500 dilution of anti-LAMP1 antibody. After incubating at room temperature for 30 minutes, remove the solution, and use a plate washer to wash the wells five times by applying 100 microliters per well of PBS.
Then, add 40 microliters per well of blocking solution containing the appropriate fluorescent secondary antibody and Hoechst 33258 at five micrograms per milliliter. Incubate at room temperature for 30 minutes. Wash the samples five times and leave the final PBS wash in the wells to keep them from drying out. To carry out image acquisition, use an epifluorescence automated microscope equipped with a 20x objective and 350, 488, 555, and 615-nanometer channels.
Acquire 21 independent fields per well in order to image a minimum of 5000 cells per sample. Apply autofocusing using the host cell nuclei channel as a reference.
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