The overall goal of the following experiment is to determine the percentage of cytosolic and vacuolar salmonella tym in infected bone marrow derived macrophages. This is achieved by infecting macrophages with M cherry expressing salmonella Ty aerium. As a second step, cells are treated with the detergent tonin, which permeables the plasma membrane, but not vacuolar membranes.
Next cells are stained with an antis salmonella antibody coupled to the Fluor ofor Zi.In order to label cytosolic salmonella ty erum, the results show the percentage of cytosolic bacteria among total bacteria based on fluorescence activated cell sorting or facts. These metals can help answer key question in the field of pathogen interaction, such as does an intracellular pathogen replicate in a VA or in the cytosol Inoculate the bacterial strain one day before the infection In three milliliters of Luria britani medium supplemented with streptomycin and ampicillin in a shaker at 37 degrees Celsius overnight to account for the individual conditions. Add cover slips to the wells of a 24 well plate that will be used as controls to plate the cells for infection seed, wild type bone marrow derived macrophages, or BM dms at a density of 0.125 million cells per well in one 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 OD 600 of the diluted culture against phosphate buffered saline. This ensures that the OD 600 is measured in the linear range of the spectrophotometer.
Calculate the concentration of salmonella per milliliter and OD 600 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 B MDMs.
Add one milliliter of macrophage medium to the uninfected control wells, B one through B three and C one through C3.Then add one milliliter of salmonella suspension to wells, A one through a five 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 a 37 degrees Celsius and 5%carbon dioxide at one hour post-infection. Remove the plate from the incubator to add 0.1 milliliter of macrophage medium containing Gentamycin to kill extracellular bacteria.
Add gentamycin 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 one milliliter of prewarm docos, modified eagles medium or DMEM.
Replace the DMEM with prewarm 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 digit toin and KHM buffer with saponin. In addition to antibody solutions as described in the text protocol to perform cell permeation, remove the plate from the incubator and wash the wells three times with 0.5 milliliters of prewarm KHM buffer.
Then remove the KHM buffer and add 0.25 milliliters of plain KHM buffer to wells A four B one and C one khm buffer with dig toin to wells, A one through A three B two and C two and khm buffer with saponin to wells A five B three and C3 incubate for exactly one minute at room temperature before immediately washing all wells three times with 0.5 milliliters of PREWARM 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 one milliliter of PBS for the infected cells in wells, A one through a five.
Prepare for fax analysis by washing the cells three times with PBS. Then add 0.5 milliliters of ice cold PBS containing 0.1%Triton. Next, transfer the cell suspension to a five milliliter fax tube with a cell strainer cap to break up cell aggregates while keeping samples on ice.
Analyze the samples on a fax machine using the fully permeable controls. Set the gate for total bacteria based on forward and side scatter and the mCherry signal. Next, analyze the fitz signal in the total bacterial population using the fully perme and not perme controls to set the gates for cytosolic bacteria and vacuolar bacteria with the gate set.
Analyze the samples and determine the percentage of cytosolic versus vacuolar bacteria using FlowJo software. According to the manufacturer's protocol for the uninfected permeation controls in wells B one through B three and C one through C3, prepare for microscopy by removing the PBS and adding 250 microliters of 4%Paraform aldehyde in PBS incubate for 10 minutes at 37 degrees Celsius following incubation. Wash the wells two times with one milliliter of PBS and at 250 microliters of 0.1 molar glycine in PBS for 10 minutes to quench the fixative, then wash the wells two times with one milliliter of PBS to perform secondary antibody staining.
Stain the permeation controls for one hour at room temperature with appropriate secondary antibodies coupled to fluoro fours in PBS. That is supplemented with 0.1%saponin and 3%BSA. To fully perme the cells wash the cover slips three times with one milliliter of PBS and mount the cover slips for analysis in mounting medium with dappy.
Analyze the cover slips with the controls at a 40 x or 63 x magnification using a confocal fluorescence microscope. Shown here are typical facts. Results positive and negative controls are used to set the gates for m cherry positive, Fitz positive bacteria, which is cytosolic, salmonella, and m cherry positive FITSI negative, which is valar salmonella.
Based on these gates, the percentage of cytosolic and vacuolar bacteria is determined in the experimental samples. In this example, m cherry positive salmonella typhimurium wild type is compared to a CA deletion mutant in bone marrow derived MI macrophages shown here is kexin and PD one staining that is obtained in permeation controls treated with digit, toin or saponin since calnexin is an er membrane protein calnexin staining can be seen throughout the cytosol and around the nucleus in both digit toin and saponin perme cells. In digit toin perme controls no PD one staining is visible.
While staining can be observed in sasin and perme cells. An anti salmonella staining result in cells that have been perme with tonin is shown here. Total bacteria are in red while cytosolic bacteria are in green.
The E positive population is salmonella with access to the cytosol. While the fite negative population is bacteria that we're residing in an intact salmonella containing vacuole or SCV and we're therefore protected from salmonella FSE labeling here, wild type salmonella is compared to a CFE deletion mutant strain. Since CA is necessary for salmonella to maintain SCV integrity, an increased percentage of salmonella is After watching this video, you should have a good understanding of how to determine the percentage of vascular and cytosolic bacteria.
