The overall goals of this protocol are to cultivate and infect C57 blacksix mice with the EGG strain of listeria monocytogenes and to measure the bacterial load, interferon gamma responses, and survival and response to infection by this pathogen. This protocol can help researchers screen for agents or genes that have an impact on interferon gamma responses and on animal survival after bacterial infection. The main advantage of this technique is that it is relatively simple to set up in the laboratory.
Even for those with little experience in cultivating bacteria. Also demonstrating the procedure will be Jennifer Ahn, a graduate student from my laboratory. Begin by dipping a sterile pipette tip into room temperature thawed glycerol stock of the L monocytogenes EGG strain.
And immediately streak the tip back and forth across a section of a brain heart infusion or BHI plate. This is the primary streak. Turn the plate 90 degrees and drag a fresh pipette tip through the first streak, spreading the stock to the next quarter of the plate.
This is the secondary streak. Then, turn and spread the secondary streak into a third quarter of the plate to make the tertiary streak. Now, turn the plate upside down at 37 degrees celsius, for an overnight incubation.
Single uniform colonies should be visible in the last set of streaks between 16 and 24 hours. When the colonies appear, dispense 10 milliliters of sterile BHI broth into a sterile vented 50 milliliter tube and use a sterile pipette tip to transfer one L monocytogenes colony from the plate into the broth. Culture the inoculated broth in an orbital shaking incubator overnight at 37 degrees celsius and 225 rotations per minute, until the OD600 equals one.
For preparation of the experimental L monocytogenes infection inoculum, in a biosafety cabinet, inoculate a tube of BHI medium with 100 microliters of the overnight culture. After another incubation at 37 degrees celsius, with shaking until the desired OD600 is reached, transfer the culture contents into a sterile centrifuge tube for centrifugation. And use a vacuum attached to a trap flask containing bleach to aspirate the supernatant.
Wash the pellet two times with PBS under the same centrifuge conditions. After the second wash, resuspend the bacteria in PBS and dilute at the appropriate concentration. To deliver the CFU of interest to each mouse in a 200 microliter volume.
Next, mix the suspension thoroughly with a sterile pipette tip to ensure that the bacteria are homogeneously distributed. Then draw 200 microliters of inoculum into a one milliliter safety engineered syringe fitted with a 25 gauge needle. Now, scruff a mouse with the less dominant hand by the loose skin around the shoulders.
And inject all 200 microliters of the inoculum in the lower quadrant of the abdomen, just lateral to the midline to avoid the bladder, using a new needle and syringe for each animal. To measure the peak infection bacterial load, on day three post infection, restrain the limbs of an experimental animal in the supine position on a dissecting board. And disinfect the skin with 70 percent ethanol.
Using sterile tough cut scissors, make an incision from the chest to the groin. Then make an incision from the mid groin toward each knee and from the mid chest toward each elbow. Blunt, dissect, and reflect back the skin, pinning it open with 25 gauge needles.
Disinfect the muscle layer with more 70 percent ethanol. Then using sterile fine scissors, make a midline incision in the peritoneal wall. Use forceps to grasp the xiphoid process.
Make incisions in the peritoneal wall starting at the xiphoid process and moving laterally on each side. Following the ribcage to just below the diaphragm. This will reveal the liver.
Next, use sterile scissors to cut an approximately 100 milligram piece out of one lobe. Place the tissue in a preweighed 1.5 milliliter micro centrifuge tube containing 0.2 to 0.3 grams of 1.5 to 2 millimeter acid washed sterile glass beads. And use the forceps to gently push aside the organs on the left side of the peritoneal cavity to visualize the spleen.
Use the forceps to gently grasp the spleen, cutting away the surrounding connective tissue to release the organ from the peritoneal cavity. Place the spleen in a different tube than the liver, and transfer the tissues to the laboratory in a leak proof container containing ice. Shake the tubes while using a bead mill homogenizer for three minutes at a 30 hertz frequency.
Then, set up a tenfold dilution series of the homogenates in 0.1 percent trident X 100 in PBS. Next, use a sterile spreader to spread 100 microliters of each diluted homogenate onto a BHI agar plate. And transfer the plates to a 37 degrees celsius incubator for an overnight incubation.
Keep the plates that contain up to 300 colonies per plate. Count the colonies on each plate. To measure CD4 in CT8 t cell interferon gamma responses, harvest the spleens from mice infected with two tenths into the fourths CFU of the pathogen as just demonstrated.
Process the spleens into a single red blood cell life suspension. And resuspend the pellet at four times ten to the six cells per milliliter concentration in complete RPMI medium containing FCS. Next, seed one milliliter of cells per well into a 24 well plate with an equal number in volume of thawed, heat killed L monocytogenes and place the cold cultures into a 37 degrees celsius incubator.
After 20 hours, add 0.66 microliters per milliliter of protein transport inhibitor to the wells. And return the plate to the incubator. Four hours later, in a biosafety cabinet, collect 500 microliters of culture supernatant and freeze the samples at negative 80 degrees celsius for the later measurement of interferon gamma by Eliza.
Then transfer the cells into a single sterile 15 milliliter tube, washing the wells with PBS and pooling the wash with the collected cells for flow cytometric staining and analysis. The peak of bacterial load occurs in the spleen two to three days post infection. And is reduced dramatically by the addition of a PPAR Alpha antagonist prior to infection.
At seven days post infection, the restimulation of spleen CD4 positive and CD8 positive t cells x vivo with heat killed pathogen elicits a positive interferon gamma response from CD4 positive and CD8 positive cells as detected by flow citometry. Treatment with the PPAR alpha antagonist prior to infection boosts the interferon gamma responses by CD4 and CD8 cells. Further, PPAR alpha antagonist treatment increases most survival after infection with the LD50 dose of the pathogen, illustrating the facility of this model for investigating the impact of new drugs that modulate interferon gamma responses on animal survival from infection.
Once mastered, all the procedures in this protocol can generate the desired data in several weeks, if performed properly. The technique can be further adapted by infecting mice with strains of listeria monocytogenes that express model antigens and using MAC class one and two tetramery agents specific for these antigens. This will allow one to measure T cell expansion post infection.
After its development, this technique became an instrumental model for deciphering the role of interferon gamma in host immunity to intracellular pathogens. After watching this video, you should have a good understanding of how to cultivate and infect C57 blacksix mice with listeria monocytogenes. And to measure the backtrail load and interferon gamma responses to infection.
Don't forget that working with listeria monocytogenes can be hazardous to immuno compromised individuals such as pregnant women, the very young or elderly. Therefore the pathogen should only be handled by immuno competent individuals under biosafety level two conditions.
