The overall goal of this procedure is to examine bacterial uptake by M cells located within the intestinal mucosal immune system. The first step of this procedure is to expose and ligate the intestinal pyres patches of an anesthetized mouse. Fluorescent bacteria are then injected into the ligated area.
An hour later, the pyres patches are excised. The cells in the tissue are then permeated and stained for the M cell marker, GP two. Finally, the samples are observed using a confocal microscope and bacteria being transcytosis by M cells can be visualized.
In addition to provide insight into the molecular basis of the bacterial transcytosis by m cells, dismissal can be applied to other system. For example, this protocol could be used to assist the cell permeability and in framed intestine using chlor and microbe Using a sterile glass spreader streak the fluorescent bacteria on an LB auger plate and incubate the plate at 37 degrees Celsius until single colonies are visible. Pick a single colony off the solid auger and inoculate it into two milliliters of fresh LB media.
Then culture the bacteria on a shaker overnight at 37 degrees Celsius. Once the starter culture has grown, transfer 500 microliters of bacteria into 4.5 milliliters of LB medium and incubate this new culture at 37 degrees Celsius for three to four hours or until its optical density at 600 nanometers reaches one indicating that the bacterial growth is in log phase. At this point, centrifuge the culture at 3000 times G for five minutes at four degrees Celsius to harvest the bacteria following centrifugation, discard the snat and wash the pellet with five milliliters of sterile PBS After repeating the wash step Resus suspend the bacteria in five milliliters of PBS.
After placing the mouse under continuous isof fluorine anesthesia use sterile tools to aseptically open the mouse's abdomen. Start by exposing the small intestine and locate the PI's patches. Once a PI's patches have been found, use sterile sewing yarn to ligate the intestine about five millimeters to one side of the patch.
Take care to avoid severing blood vessels during the procedure. Once the intestine is ligated, use a syringe to inject 50 microliters of bacterial suspension about 10 to the seven CFU into the ligated pyres patch loop on the loose side of the intestine. Then use yarn to ligate the other side of the intestine.
Close the mouse's abdomen with a clip and maintain the mouse under anesthesia for one hour before euthanizing it. To harvest the pyres patch, carefully excise the pyres patch. Then vigorously flush PBS through the harvested section of intestine several times to wash the apical side of the PI's patch and remove excess mucosal fluid and bacteria.
Once the PI's patch is washed, place it in a well of a 24 well plate and add cyto effects or cyto perm solution. Incubate the sample on ice for one hour. Once the P'S patch is fixed, soak it in one milliliter of perm wash buffer for five minutes.
Repeat this wash step three times. Then place the sample in one milliliter of blocking buffer and incubate it on ice for 30 minutes. Following the blocking step, add anti mouse GP two monoclonal antibody to a final concentration of five micrograms per milliliter and incubate the sample overnight at four degrees Celsius to stain the M cells.
Next, wash the sample as before. Add a secondary floor for conjugated antibody and incubate the sample on ice in the dark for two hours. Once the sample is stained, wash the sample gently in PBS.
Then place three or four pieces of circular cover glass on a slide and embed the pires patch in 200 microliters of a 30%glycerol solution in PBS. On the slide, use a DM IRE two confocal laser microscope, or a Delta vision restoration deconvolution microscope to observe the sample. In this image, green GFP labeled salmonella typhimurium can be seen to be surrounded by GP two shown in red on the apical plasma membrane.
In this rotated image, bacteria are visible within the sub apical cytoplasmic vesicles. After watching this video, you should have a good understanding of how to assess bacterial transcytosis by M cells using ligated pys intestinal Lu asay.
