This video demonstrates a method for measuring bacterial load and generating and analyzing single cell suspensions from mouse, liver, and spleen. First, a listeria inoculum is prepared and injected intravenously into mice. Blood, liver and spleen are then harvested from euthanized mice at chosen time points.
Single cell suspensions are prepared from harvested tissues and tissue homogenates AC cultured on horse blood agar plates. The resulting cell suspensions can then be analyzed for viability and immune cell content by fax analysis while the bacterial load is determined based on colony forming units. This method can help characterize immune responses to steroid infection, such as those between susceptible and resistant mal strains while simultaneously determining bacteria load in infected organs.
Begin this procedure by preparing the listeria inoculum to the desired concentration instructions for culturing, storing and preparing listeria can be found in the accompanying written protocol. Once the inoculum is ready, transfer two aliquots of 100 microliters to sterile micro fused tubes containing 900 microliters of PBS to make a one to 10 dilution, then transfer 100 microliters of each dilution to horse blood agar HBA plates and spread thoroughly with a sterile spreader for performing colony forming Unit CFU counts then incubate the plates inverted at 37 degrees Celsius overnight. Next, transfer the mice to a clean cage.
Place the cage 50 centimeters under a 250 watt infrared lamp for five minutes to ensure that the tail vein is dilated. This allows for better visualization during the injection here, the listeria resistant mouse strain, C 57 black six, and the listeria susceptible mouse strain. Valve C will be injected gently mix the listeria suspension by inverting the tube a few times.
Then load a one milliliter syringe with a 27 gauge needle for injection of 200 microliters per mouse. Ensure that any air bubbles within the syringe are bled out prior to injection. Place the mouse in a restraining device.
Then locate the lateral tail vein of the mouse by first identifying the tail artery, which is found in the middle of the dorsal side. Then turn the tail slightly to the lateral side to identify the vein. Gently wipe the tail with a 70%ethanol wipe then inject after withdrawing the syringe.
Briefly apply pressure to the entry wound with a sterile gauze square. Note that it's much more difficult to identify the tail vein in black colored mice following the injection. Return the mouse to its cage after injecting all of the mice plate and incubate the remaining inoculum for colony forming unit counts.
As before the next day, count the number of colonies and determine the pre injection and post-injection concentrations of the listeria inoculum. The characteristic halo surrounding individual colonies as shown here is due to beta hemolysis. Once the pre and post-injection counts have been determined, calculate the average colony forming units by adding the pre and post-injection colony forming unit counts and dividing by two.
Finally, at various time points following injection, collect blood and harvest the liver and spleen from euthanized mice. If fax analysis will be performed, the liver must be perfused before removal to obtain blood, perform a cardiac bleed immediately after euthanasia and collect as much blood as possible in an appropriate collection tube. To perfuse the liver, first, open the abdominal cavity and remove and discard the gallbladder.
The interior vena caver is a vein that runs parallel to the spine and transports blood from the lower part of the body, including the liver into the heart. Using a pair of scissors, serve the vein just below the heart to create an outlet for the perfusing fluid to exit. Next, flip the liver lobes towards the head and push the intestines to the right to expose the hepatic portal vein, which feeds into the bottom of the liver.
From the lower right hand side, insert a bent half inch length 26 gauge needle in the hepatic portal vein and inject 10 milliliters of PBS. After one to two milliliters of PBS has been injected, the liver will turn from a dark red color to light brown. The injected solution will exit from the severed inferior vena caver Liver perfusion ensures that harvested cells are from the liver and not the circulating blood.
Next, remove the perfused liver from the mouse's body cavity and submerge it in the tube of facts buffer on ice. Then remove the spleen and place it on ice for processing in a similar manner as described in the next section. For the liver to generate single hepatic cell suspensions, begin by transferring the perfused liver from fax buffer to a 70 micron cell strainer placed inside a 60 millimeter Petri dish.
Using a five liter syringe plunger, push the tissue through the cell strainer into the Petri dish. Transfer the single cell suspension to a 50 milliliter conical screw cap tube and bring the total volume to 40 milliliters. With cold fax buffer.
Vortex the cell suspension. Then set a one milliliter quata aliquot aside for determination of bacterial load. Later in the procedure, place the 50 milliliter tube in the centrifuge and pellet the cells by spinning up 500 times G for five minutes of four degrees Celsius.
After discarding the supinate, repeat this wash step by Resus suspending the pellet in 40 milliliters of cold fax buffer and centrifuging. Again, resus. Suspend the pellet in 20 milliliters of isotonic per hole at room temperature centrifuge the cell suspension at 700 times G for 12 minutes at room temperature.
Following the spin, a disc like sheet of hepatocytes will float on top of the perol and the leukocytes will rep pelleted at the bottom of the tube, discard the hepatocyte layer and perol by pouring it into a waste container. Then reus suspend the leukocyte containing pellet in four milliliters of TAC buffer to Liz Erythrocytes and incubate the cells at room temperature for up to 10 minutes with frequent inversion. Following the incubation, filter the cell suspension through a 100 micron nylon membrane into a new 10 milliliter centrifuge tube.
Underlay the filtered cell suspension with one milliliter of fax EDTA buffer as shown here. Then centrifuge at 350 times G for five minutes of four degrees Celsius. After the spin, resus bending five milliliters fax EDTA buffer, then spin again and resend the pellet in 0.5 to three milliliters of fax, EDTA buffer, depending on the size of the pellet, determine the number of viable hepatic leukocytes using a hemo cytometer based on triam blue exclusion liver.
Single cell suspensions can then be labeled with antibodies specific for desired cell surface markers and analyzed by facts. Once single cell suspensions have been generated from liver and spleen tissue, bacterial loads will be determined for parts of the procedure. A stomach co machine will be used to homogenize the tissue holding a stomach ER bag shut to prevent leakage.
Begin by laying it flat on a clean bench. Roll a thick round pen over the tissue until the tissue is mashed within the bag. Add five milliliters of PBS to each stomach co bag containing mashed tissue.
Place the stomach or bags into the stomach and run on high speed for 10 minutes. The stomach machine has two paddles which press the stomach or bags at a specified speed, blending the contents into a uniform homogenate and containing any aero aerosols using a pipette, mix the homogenate in the bag. Then transfer 300 microliters in duplicate to a 96 well flat bottom plate.
Within the wells of the 96 well plate perform one to 10 serial dilution to a 10 to the five dilution. For each tissue homogenate sample, carefully use a sterile spreader to spread 0.1 milliliters of each dilution onto a pre dried HBA plate. Incubate plates of 37 degrees Celsius overnight.
The next day, count the number of colony forming units for each dilution and calculate the colony forming units per tissue taken into account the portion of the tissue dilution factors and volume of homogenate. See if C 57 black six mice were infected with around 2000 colony forming units of listeria at three and seven days post-infection, mice were euthanized and the liver was perfused and harvested with the spleen. Dilution of splenic homogenate or hepatic single cell suspension were cultured on HBA plates to determine the bacterial load.
In the results shown here, the dotted line indicates detection limit at 100 colony forming units per organ. Note that the listeria load is higher at day three than day seven. This is because by day seven, various immune cells lead to the clearance of listeria.
In these two tissues, spleen and liver, single cell suspensions were stained with trian blue and loaded onto a hemo. Cytometer cells within the counting area indicated in the red box will be counted. Non-viable cells will take up triam blue and appear blue, whereas viable cells will remain unstained.
As shown here, the number of viable cells in the spleen shown on the left and the number of viable leukocytes in the liver shown on the right increases during the course of infection. This increase is due to immune cells, including neutrophils and lymphocytes that become activated, proliferate and migrate to these two sites of infection. As an example of immune cell levels during the course of listeria infection, representative fax plots and the number of CD eight positive T cells are shown here.
The number of CD eight positive T cells transiently decreases due to lymphopenia in the spleen at days two to three before increasing noticeably from day five post-infection in both the spleen and liver. Following this procedure, other methods such as oscillation of androgens, antigen specific T cells and functional assays of sorted immune cells can be performed to further characterize immune responses to listeria infection.
