The overall goal of this procedure is to establish and maintain a natural hain infection model. First mice are infected with tra curus mirrors eggs by oral gavage. 21 days later, the infected animals are euthanized and blood mesenteric, lymph nodes, fecal pellets, colons, and seeker are harvested.
Using these samples, the immune response generated against tricks. Mirrors can be analyzed. Serum samples can be analyzed by lysa to detect TRA specific antibodies.
In vitro restimulation of lymph node cells can be performed to analyze cellular immune responses. Western blotter fecal pellets can be used for protein content assessment and gene expression analysis can be performed by QPCR analysis of the colon. Finally, the cecum tip can be used for histology while the rest of the cecum may be used to determine the worm burden in infected mice.
Ultimately, results can be obtained that show if a mouse is resistant or susceptible to trais MOUs infection and the immune response mounted against the parasite can be characterized. This method can help answer key questions in the immunology field, such as which factors control T helper cell activation, as well as which proteins are required for TH two cell differentiation. Trauss MOUs eggs can be propagated in immunodeficient mice or genetically susceptible strains such as a KR 35 days after infection to harvest eggs from euthanized mice.
Expose the animal's ventral surface and rinse the mouse's abdomen with ethanol using sterile forceps and scissors. Grasp the abdominal skin and make a small incision. Expose the abdominal contents, identify the cecum and the proximal colon, and then remove them.
Transfer the organs into a 10 centimeter Petri dish containing penicillin and streptomycin, supplemented DMEM. Then cut the cecum and proximal colon open, exposing the lumen and worms using forceps. Place the open organs in a beaker containing PBS and gently shake to wash off feces.
Once the cecum has been washed, transfer it to a fresh Petri dish and use smooth curved forceps to gently pull out the worms. Transfer all the worms from each mouse into one well of a six Well plate containing five milliliters of antibiotic supplemented DMEM. Place the six well plate with a damp paper towel inside a Tupperware container and incubate at 37 degrees Celsius for four hours to allow the worms to lay eggs.
Following this incubation, use forceps to remove the worms from the original well and split them into two new wells containing DMEM. Splitting the worms in this manner will maximize egg recovery using a pipette. Harvest the remaining medium containing secreted antigen and eggs from the original well and transfer it to a 15 milliliter Falcon tube.
Centrifuge the tube at 3000 RPM for five minutes. Then remove and save the supinate containing four hour antigen and Reese's bend the egg containing pellet in sterile water. Place the six well plate containing worms at 37 degrees Celsius once more and incubate it overnight the next morning.
After discarding the worms, harvest the contents of the wells as shown earlier and centrifuge the supernatant to isolate the eggs. Save the supernatant containing overnight antigen and resuspend the eggs in water. As before, combine these eggs with the eggs obtained from the four hour incubation.
Filter the eggs through a 70 micron strainer to remove any leftover worms. Then transfer them into a 75 centimeter square flask. Cover the flask in aluminum foil to shield it from light and store the eggs for six weeks at room temperature.
This will allow the eggs to mature and embry anate after six weeks. Wash the eggs and stir our water and count viable eggs. Seen here.
As an example of a viable embryonated egg and a non-viable egg, Reese's bend the Exeter concentration of 50 per 50 microliters. At this point, the eggs may be stored at four degrees Celsius before infecting mice. Be sure to thoroughly resend the egg containing solution.
Transfer the desired amount of inoculum into a 14 milliliter snap cap tube and invert the tube to mix. Draw up 200 microliters of eggs into an appropriate size feeding needle for animal size fitted to a one milli liter syringe. Scruff the mouse and administer the eggs by oral gavage.
Return the infected mouse to its cage and allow the infection to proceed for 21 days. After 21 days, use a non heparinized syringe to harvest the blood of infected mice by cardiac puncture. Following carbon dioxide euthanasia, the blood samples can be stored at four degrees Celsius.
For serum isolation and analysis of tra specific IgGs and IgE by Eliza. Expose the mouse's abdominal cavity as described earlier. Then use forceps to pull out the cecum with scissors or another pair of forceps.
Push the small intestine over to the right, exposing the mesenteric lymph nodes. Gently remove the lymph nodes while being careful not to cut into the intestines and place them into a 15 milliliter Falcon tube containing two milliliters of media. Lymph node cells can later be reed in vitro with four hour antigen S supernatant from the worm cultures to analyze the cellular immune response to the parasite.
Once the lymph nodes have been harvested, excise the secum and colon as described earlier, using forceps, liberate fecal pellets from the distal colon, placing them in the two milliliter oxygen tube pellets can be stored at minus 20 degrees Celsius and analyzed for protein content by western blot using scissors. Cut approximately one centimeter of proximal colon and place in a tube containing RNA. Later store at four degrees Celsius.
Cut off about five millimeters from the tip of the cecum holding the tip of the cecum with forceps. Use a three milliliter syringe and a 22 gauge needle to flush the sample with PBS. Then transfer it to a five milliliter flk and snap cap tube containing 4%paraldehyde.
The paraldehyde fixed cecum sample can be stored at four degrees Celsius and used for histological analysis. Place the rest of the cecum in a Petri dish and freeze it at minus 20 degrees Celsius to kill worms. For easier enumeration to enumerate worms, thaw the secum and place it in a Petri dish containing about five milliliters of water using scissors.
Cut the secum open to expose the lumen and shake to collect feces into a dish. Place the clean secum into a fresh Petri dish containing water and use smooth curved forceps to scrape off the intestinal epithelial cells. Transfer the secum to a new Petri dish containing water and rip the tissue up into small pieces.
E observe the feces, intestinal epithelial cells, and cecum tissue under a dissecting microscope and enumerate the worms, removing them as they're counted and placing them in a fresh dish. This is how the worms in the cecum should look before and after isolation as shown here. Following tragus mirrors infection black six are able to control the parasites while a KR mice develop elevated parasite burdens.
Similarly susceptible A KR mice develop markedly higher TRA specific IgG two a titers than black six mice as determined by serum Eliza. In this experiment, Western blot analysis revealed that the amount of goblet cell specific protein realm beta was increased in the feces of black six, but not a KR mice following infection. Histological analysis of the distal cecum shows goblet cell hyperplasia and mucus production in resistant TRAs MOUs infective black six mice, but not in susceptible a KR mice.
After watching this video, you should have a good understanding of how to propagate trismus eggs, infect experimental mice, and harvest the intestinal tissues, meso, enteric lymph nodes, and blood for analysis of infection in susceptible or resistant mice.
