The overall goal of the following experiment is to investigate lung inflammation and immune cell responses. This is achieved by purifying helminth eggs from shta infected livers. As a second step, mice are immunized and challenged with purified eggs, which establishes a potent lung inflammatory response.
Next, lungs and draining lymphoid organs from the mice are isolated in order to assess the magnitude of the inflammatory response to the helmet. Results obtained show potent hel induced lung inflammation based on recruitment of inflammatory cells to the lung and observation of the egg induced lung pathology. Generally, individuals new to this technique will struggle a little because care precision is needed for dissection of the lung and lymph nodes as well as injection of the eggs.
However, perfecting this technique is rewarding because it allows visualization of a potent inflammatory lung response to the helmuth eggs. Demonstrating the technique are Karen and will technicians in the Nyer and Greenberg laboratory Place a euthanized mouse that has been infected with Schistosoma circ on its back and an incision through the skin to expose the peritoneum, excise the liver, which is located below the rib cage and diaphragm mince the livers finely and add 20 milliliters per liver of digestion mixture, which consists of collagenase, dys, base, penicillin, and streptomycin in PBS. Place this mixture in a 50 milliliter Falcon tube and incubate overnight in a 37 degrees Celsius shaker.
The next day. Centrifuge the mixture. Pour off the excess fluid and fill the tube with PBS centrifuge again and repeat the PBS wash after the last centrifugation.
Resuspend the pellet in 25 milliliters of PBS. Next strain the resuspended pellet through a metallic strainer, followed by passing through a sieve. Place this strained mixture in a 50 milliliter Falcon tube and centrifuge.
Pour out the supernatant and resus. Suspend the pellet in three milliliters of PBS. Overlay the mix gently onto a 40 milliliter gradient of eight milliliters per call and 32 milliliters of 0.25 molar sucrose centrifuge for 10 minutes at 800, G at 20 degrees Celsius.
Next, discard the top layer of liver cells using a transfer pipette. Remove the egg pellet at the bottom and place it in a 50 milliliter falcon tube. Wash the pellet three times in 10 milliliters of PBS containing one EDTA and EGTA and centrifuge.
Resuspend the pellet in 500 microliters of PBS. Overlay this mixture on a new 10 milliliter gradient containing 2.5 milliliters of per call and 7.5 milliliters of 0.25 molar sucrose and centrifuge. Wash the pellet three times in 10 milliliters.
PBS and centrifuge. Make sure the Falcon tube is mixed well and remove 50 microliters from the final wash. To count the eggs dilute the 50 microliter.
Mix with 450 microliters of PBS and add 100 microliters of this mixture on a glass slide on a dissecting microscope stage to count the droplets resuspend the eggs at 50, 000 eggs per milliliter in PBS eggs can be stored for several months at minus 80 degrees Celsius and thawed once or twice for use. Before use. Inspect the eggs under a microscope to ensure they're still intact in a five milliliter snap cap tube.
Prepare the eggs at 5, 000 eggs per 100 microliters of PBS for injection. Estimate 50%more eggs than necessary. Load a 23 gauge, five eighths of an inch needle and a one milliliter syringe with 100 microliters of egg suspension per mouse.
Rock the syringe back and forth to mix eggs prior to each injection and inject 100 microliters of suspension per mouse intraperitoneal Ely 14 days after sensitization. Prepare eggs as before at 5, 000 eggs per 100 microliters and load a one milliliter syringe and 23 gauge needle with eggs. Use forceps to bend the needle at a 90 degree angle and ensure no bubbles are present.
Anesthetize the mouse and place it on its side. Inject 100 microliters of suspension into the vessels behind the eye at a 45 degree angle to the nose. Eight days after the intravenous challenge, euthanize the mouse and make a small incision in the skin of the abdomen.
Cut open the peritoneum and locate the abdominal aorta. Cut the aorta in order to recover blood with a pasture pipette and store this on ice. Move the liver down to expose the diaphragm.
Use fine scissors to cut the diaphragm and carefully cut. Open the rib cage to expose the lung tissue. The para thymic lymph nodes are located underneath the rib cage above the lungs and on either side of the thymus.
Recover the lymph nodes with fine forceps and store them on ice in one milliliter of sterile media. To begin the intubation procedure, move the salivary glands to the side to expose the trachea. Place a fine forceps under the trachea and using fine scissors.
Cut a hole in the middle of the trachea and insert tubing into the lungs. Secure the tubing by tying it with surgical thread. Now attach a PBS filled one milliliter syringe to the tubing and inflate the lungs with one milliliter of PBS.
Carefully retrieve the BAL wash with the syringe and store this on ice. Next, attach a 4%para formaldehyde in PBS filled one milliliter syringe to the tubing and inflate the lungs. Then remove the tubing and tighten the surgical thread to prevent the PFA from leaking.
Finally, carefully dissect out the lung tissue and place it in a 50 milliliter falcon tube with five milliliters of 4%PFA in PBS. Following a 0.5 to two hour incubation on ice centrifuge collected blood to separate serum layer serum can be used for S mansoni egg antigen specific IgG isotype Eliza's excised paraic lymph nodes can be re stimulated with s mansoni egg antigen to analyze antigen specific TH two cytokines following centrifugation and pelleting cells recover BAL fluid for analysis of TH two cytokines by Eliza. BAL cells are recovered for cyto centrifuge preparation.
Overnight fixed lung tissue for histological and immunofluorescence lysis shown here are pictures of granulomatous livers, which are characteristic of high esman egg burdens. Also pictured are esman eggs. This figure represents airway inflammation driven by injection of essman sooni eggs.
The graph on the left shows that BAL cell numbers are much higher in s mansoni egg injected mice when compared with naive mice. The images on the left are representative cyto centrifuge. Preparations of BAL cells and the inflammatory infiltrate consisting of macrophages, eosinophils, and lymphocytes can clearly be visualized here.
A pulmonary granuloma surrounding the esson egg is shown in an H and E stained lung section. This image depicts immunofluorescent staining for realm alpha shown in green. The mano receptor shown in red and DPI dye shown in blue, which reveals alternatively activated macrophages seen by the white arrow.
The S mansoni egg antigen specific TH two cytokine response in the paraic lymph nodes of naive and S mansoni injected mice are shown here. Lymph node cells from each group were stimulated with s mansoni egg antigen for 72 hours. And then Eliza of the supernat was performed for IL four IL five and IL 13.
Concentrations of all three inflammatory cytokines were much higher in the esman injected mice After its development. This technique provides an easy way for researchers in the field of allergy and helmet infection to investigate immune cells recruited in response to infection with parasitic helmets and allergy in a mouse model organism.
