The overall goal of the following experiment is to isolate intact legionella containing VAEs or LCDs from infected phagocytes. This is achieved by first homogenizing the infected phagocytes with a stainless steel ball homogenizer. During the second step, the homogenate is incubated with a primary antibody that specifically targets a bacterial effector protein localizing to the LCV membrane, followed by incubation with a secondary antibody coupled to magnetic beads.
Next, the antibody decorated lc vs. Are retained by a magnetic field and then washed and alluded. Ultimately, the magnetically enriched LCDs can be further purified by density gradient centrifugation.
The main advantage of this technique over existing methods is that compartment specific molecular characteristics of the infected parasites can be exploited to purify intact pathogen bacterias. Four days before LCV isolation streak out DS red express producing legionella pneumophila from glycerol stocks on charcoal yeast extract auger plates with five micrograms per milliliter. Chloramphenicol incubate the bacteria at 37 degrees Celsius one day before infection CDA one times 10 to the seventh Calnexin GFP producing Dium Disco Dium in 75 square centimeter tissue culture flasks incubate the amoeba in 10 milliliters of HL five medium with G four 18 at 23 degrees Celsius on the day before the experiment inoculate a 15 milliliter test tube containing three milliliters of a YE medium and five micrograms per milliliter of chloramphenicol with approximately 100 milliliters of an L pneumophila suspension.
To yield an optical density at 600 nanometers of 0.1, incubate the overnight culture on a rotation wheel at 37 degrees Celsius for 21 to 22 hours before beginning the isolation. First, change the medium of the dedis qadium cells to remove the antibiotic. Now measure the optical density of the one to 10 diluted L pneumophila overnight culture, which should be around 0.3.
Then transfer approximately 500 microliters of the L pneumophila suspension to each flask for infection of the dsco Dium cells synchronize the infection by centrifuging the bacteria onto the cells for 10 minutes at 500 times G and 25 degrees Celsius, followed by an hour. Incubation of the dqu dium cells at 25 degrees Celsius. After the infection.
Remove the medium and wash the ameba once with ice cold source C buffer. To remove any extracellular bacteria, add three milliliters of HS buffer supplemented with protease inhibitors to each flask, and then use a cell scraper to collect the cells. Homogenization of the infected cells is the trickiest part of the procedure.
Success depends on a meticulously clean stainless steel ball homogenizer the correct exclusion size of the ball homogenizer an odd number of strokes and the amount of pressure applied. After pooling the corresponding samples in a 15 milliliter test tube, wash a stainless steel ball homogenizer bearing an eight micron clearance ball with distilled water, and then flush the homogenizer with ice cold hs buffer. Now fill a three milliliter plastic LOR lock syringe with the first three milliliter aliquot of the cell solution and mount the syringe onto the homogenizer.
Press the sample back and forth nine times through the homogenizer and then collect the homogenized sample in a new 15 milliliter test tube. Discard the used syringe and mount a new one. After pooling the entire homogenized sample, transfer a 150 microliter Eloqua into an einor tube for later analysis.
Then block the homogenate with 2%calf serum for 30 minutes on ice on a shaker. After blocking vortex, a primary antibody against any bacterial marker that exclusively binds to the LCV membrane and then incubate the sample with the antibody on ice on a shaker for one hour. In the meantime, add 5.75 milliliters of a 35%histo den solution to a fresh 15 milliliter conical tube.
Carefully overlay 5.75 milliliters of a 10%histo den solution and then lay the tubes horizontally for one hour at room temperature. Now pellet the blocked and antibody treated homogenate for 15 minutes at 600 times G and four degrees Celsius. After discarding the supernatant, resuspend the pellet in 1.5 milliliters of HS buffer, and then transfer the sample to a fresh 15 milliliter test tube.
Next, vortex the secondary antibody and then incubate the homogenate with the secondary antibody and then place the sample onto ice on a shaker for 30 minutes. In the meantime, place the appropriate column on the max magnetic holder and equilibrate the column with half a milliliter of HS buffer. After 30 minutes, apply the sample to the column watching the column three times with hs buffer.
Then remove the column from the magnet and allude the lc vs. With 0.5 milliliters hs. Buffer by immediate plunging.
Now slowly turn the histo dens gradient tubes back to the vertical position and carefully load the elu on top. Centrifuge the tubes for one hour at 3, 350 times G and four degrees Celsius. Finally, use a long glass pasture pipette to collect eight 1.5 milliliter fractions from the bottom of each 15 milliliter tube and place the aliquots into eph tubes.
Transfer 150 microliters of each continuous density gradient fraction into EPI DPH tubes for later analysis. Begin by placing poly L lysine coated cover slips into each well of a 24 well flat bottom tissue culture plate. Pipette the samples put aside during the LCV isolation into the wells and add 0.5 milliliters of HS buffer to dilute the high histo ends concentration centrifuge.
The 24 well plate for 10 minutes at 600 times G and four degrees Celsius. Carefully remove the supernatant and then fix the samples with 4%pair of formaldehyde for 20 minutes at room temperature after fixation, wash the samples twice with source C and then mount the samples on glass slides with the appropriate mounting medium. Finally, analyze the samples with a fluorescent microscope equipped with the appropriate filters.
This first figure shows a representative overview of samples collected during the LCV isolation from dsco, dium, or macrophages ages. The homogenate of L Pneumophila infected phagocytes contains intact lc vs. But also a lot of cell debris and extracellular bacteria after pelleting.
The cell solution. The reserve sample content is similar to that of the homogenate, although sometimes a bit more dense as seen here. The flow through of the magnetic column contains mostly extracellular bacteria and cell debris since intact lc vs.
Should stick to the column after eluding the sample from the column. The eluate contains large amounts of intact lc vs. After histo dens density centrifugation.
The LCV purification appears to be more effective for dsco dium than for macrophages with dsco dium. The pathogen VAEs appear more round and the yield of isolated l cvs is more than 10 times higher compared with macrophages in intact macrophages lc vs. Stained with different antibodies also appear more irregularly Following risk procedure.
Other methods like proteomics or immunofluorescence microscopy can be performed in order to characterize in detail host cell and bacterial components of STDs.
