The overall goal of the experiment demonstrated in this video is to assess whether the antimicrobial peptide lyin plays a role in the amelioration of dextran sulfate induced colitis in mice. This is achieved by injecting bone marrow from wild type mice into irradiated licit and knockout mice and bone marrow from knockout mice into irradiated wild type mice. The transplanted bone marrow will generate new blood cells in the recipient of donor genotype.
The recipient mice are then exposed to dextran sulfate to induce colitis after four weeks. Successful transplantation is validated by flow cytometry and colonic tissue damage is assessed by histology. Results are obtained that show colitis is ameliorated when wild type bone marrow is transplanted into knockout mice.
Conversely, colitis is worsened when knockout bone marrow is transplanted into wild type mice. This method can help answer key questions in determining the gene expressions from bone marrow derived cells or non bone marrow derived cells in inflammation or infection. C 57, black six, wild type, and C elicited knockout mice are recommended for this experiment to make it possible to track the wild type mice and knockout mice.
Donor cells after bone marrow transplantation. Use CD 45.1 mice to represent wild type mice and CD 45.2 for the knockout mice. The chart shown here describes the experimental setup five donor mice and 10 recipient mice are assigned for each group.
For groups A and B bone marrow will be transplanted from the wild type mice to the knockout mice. Colitis will be induced in group A, but not group B.For group C and D.Bone marrow will be transplanted from the knockout mice to the wild type and colitis will be induced in group C only for E and F.The transplant will be from wild type to wild type and for groups G and H, the transplant will be from knockout to knockout. To prepare donor bone marrow for transfer to irradiated mice, begin by extracting bone marrow from the bones of sacrificed mice in a biosafety cabinet.
Use sterile dissection instruments to expose the humerus, femur, tibia, and fibula bones free from attaching muscles and ligaments and transfer them to a Petri dish. Cut open both ends of bones to show the red bone marrow. Then grasping one of the bones with forceps.
Flush the red bone marrow out with cold PBS with three milliliter syringe and 25 gauge needles to another petri dish. Then flush the bone from the other end to yield more bone marrow cells. Repeat this process with each of the bones.
Pool the bone marrow of wild type and knock out mice separately. Use a six milliliter syringe with an 18 gauge one half inch needle to break red bone marrow plugs by repeated aspiration and ejection. Then use a serological pipette to transfer the collected bone marrow to a 50 milliliter conical tube.
Once all of the bone marrow has been transferred, spin the bone marrow down at 740 G for five minutes at four degrees Celsius. After the spin, discard the supernatant and resuspend pellet by vortexing for five to 10 seconds. To the pellet, add five milliliters of one XRBC lysis buffer per donor mouse vortex again for five seconds and incubate for exactly three minutes at room temperature.
After three minutes, add 20 milliliters of cold PBS to each tube and mix by inversion to stop the lysis. Then pour the contents through a 40 micron cell strainer into a new 50 milliliter conical tube. Rinse the original tube with five milliliters of PBS and transfer the solution to the cell strainer.
Continuing to collect the flow through in the conical tube. Then bring the volume up to 50 milliliters with PBS and mix by inversion. Next, determine the viable cell count by trian blue exclusion using a hemo cytometer.
Once the cell count has been determined, spin down the bone marrow cells at 913 times G for five minutes at four degrees celsius. After the spin, remove the supernatant based on the total cell counts. Resus suspend the pellet in PBS at one times 10 to the eighth cells per milliliter.
The bone marrow cells are now ready for transfer into irradiated recipient mice. The recipient wild type and knockout mice for this experiment have been irradiated with 1000 rad as described in the accompanying document, the mice are bred and housed in a pathogen free facility four to 24 hours after irradiation warmed the recipient mice on a heating pad under a warming lamp to inject the donor bone marrow. Place a recipient mouse in restrainer, wipe the tail with 70%ethanol to sterilize the injection site and promote local vasodilation for easy injection.
Using an insulin syringe with a 28 and a half gauge needle, inject one times 10 to the seventh cells in one to 200 microliters into the tail vein of each mouse following injection. Place the mouse in a cage with up to three other injected mice for the first four weeks following injection. Maintain the injected recipient mice in autoclave cages with water containing the antibiotic sulfa trim.
This allows time for the animals to regain immunity, change cages, antibiotic treated water and food every four days to maintain hygiene. Four weeks after irradiation and bone marrow transplantation, switch to regular water without antibiotics and maintain the mice for two more weeks to regain their normal gut microflora. Six weeks after bone marrow injection, measure initial body weight to induce colitis.
Give the mice in the colitis experimental group 5%dextran sulfate in drinking water. Five days later, pool the peripheral blood from four mice in a vacutainer heparin coated tubes and place it on ice. Then in a 1.5 milliliter micro centrifuge tube, combine 300 microliters of blood with 300 microliters of cell staining, buffer and aliquot the blood into five flow cytometry Test tubes labeled one to five each with 100 microliters on ice.
After sacrificing the mice, measure the body weight again and calculate the change. Dissect colon tissues and pace it in formalin for h and d staining. Then obtain bone marrow cells as before.
Divide the resuspended bone marrow into five micro centrifuge tubes labeled one to five each with 100 milliliters. In preparation for analysis by flow cytometry, prepare the antibody mixture for each respective tube in the dark as described in the accompanying document, blood and bone marrow samples labeled number one will serve as controls and will not be stained blood and bone marrow samples. Labeled number two will be stained with fitzy isotype control PE isotype control and CD 16 slash 32 blocking solution.
These isotype controls are used to confirm specificity of primary antibody binding samples. Labeled number three will be stained with FZ CD 45.1 and CD 16 slash 32. Blocking solution samples labeled number four will be stained with CD 45.2 and CD 16 slash 32.
Blocking solution. Single color labeling can reveal the presence of donor or recipient cells in the blood or bone marrow. Clearly samples labeled number five will be stained for CD 45.1, CD 45.2 and CD 16 slash 32.
Blocking here the dual color labeling show relative ratio of donor and recipient cells in the blood or bone marrow. Add five microliters of antibody mixture two and five to their respective sample tubes. Then add three microliters of antibody mixture three and four to their respective sample tubes.
Incubate all of the tubes on ice for 30 minutes. Following the incubation, add two milliliters, one XRBC lysis buffer to each tube. Then incubate the samples for another 15 minutes on ice in the dark.
Spin down the cells at 350 times G, five minutes at four degrees celsius. After the spin, remove the supernatant resuspend the pellet with 500 microliters cell staining buffer in the dark. Then vortex briefly.
Next, perform flow cytometry on the immuno stain blood and bone marrow samples. Select FZ to represent wild type CD 45.1 and PE to represent knockout CD 45.2. Analyze the flow cytometry results using Flo Joe's software.
Calculate the ratio of fit E to PE ratio or PE to fit e ratio to determine whether the donor genotype is dominant genotype in blood and bone marrow. This image shows sample h and e images of colons with normal histology and DSS colitis. Histology images are scored based on mucosal tissue damage and neutrophil infiltration into mucosal and submucosal tissues.
Thus, a higher histology score indicates more severe colitis to test whether lyin plays a role in the prevention of colitis. Bone marrow was exchanged between wild type and knockout mice as described in this video. Histology was then evaluated and scored as shown here.
Histology scores were significantly increased when knockout mouse bone marrow was introduced into irradiated wild type mice. Conversely, the scores were significantly decreased when wild type bone marrow was introduced to knockout mice. These data indicate that endogenous C from bone marrow derived cells has anti-inflammatory effects and ameliorates colitis to verify that transplantation was successful in these mice.
Flow cytometry was also performed on bone marrow and blood from the mice following transplantation. Wild type bone marrow stains strongly for Fitz CD 45.1 but not pe, CD 45.2 as shown here, knockout bone marrow stains strongly for PE but not Fitz as shown here. Following transplantation, however, both bone marrow and blood from wild type mice that received knockout bone marrow stained strongly for PE but not Fitz, indicating that the predominant population of stained cells was from the donor.
Likewise, bone marrow and blood from knockout mice that received wild type bone marrow stained strongly for fitzy but not pe again confirming that the predominant stained populations were of donor origin. After watching this video, you should be able to perform bone marrow transplantation experiment and should be able to modify colitis via bone marrow derived cells.
