The overall goal of this procedure is to compare the tissue specific migratory abilities of two or more cell subsets in the same mouse recipient, allowing the analysis of many tissues and cells in one experiment. Here we show a method for comparing the migratory abilities of in vitro generated gut tropic and control T cells in a wildtype recipient mouse. The method can be used for example, to determine the ability of gut tropic T cells to migrate to the intestine mesenteric lymph nodes, peripheral lymph nodes, and spleen.
Compared to control T cells, first gut tropic and control effector T cells must be generated in vitro. Next, the experimental and controlled T cells must be labeled with differential dyes and mixed in a one-to-one ratio. Then the cell mixture, except for a small Eloqua used for confirming the input ratio is injected into the recipient mouse.
Finally, the mouse is sacrificed 18 hours after the adoptive transfer and the organs of interest are analyzed for the presence of gut tropic and control T cells by flow cytometry. The main advantage of this technique over existing methods like adopted transfer of single cell population is that a combination of different subtype is injected in the same recipient mice greatly reducing the variability associated with intravenous injection or from using different recipient mice. The implication of this technique extend to our therapy of diseases associated with massive leukocyte infiltration such as inflammatory bowel disease.
For instance, it allow us to determine ho many molecules require during normal as well as during aran lymphocyte migration to the intestine. Furthermore, this procedure allow us to assess the efficacy of drugs designed to block leukocyte trafficking Code each well of a 24 well plate with 500 microliters of PBS containing 10 milligrams per milliliter, anti CD three and anti CD 28 antibodies. Incubate the plate for two hours at 37 degrees Celsius in 5%carbon dioxide while the wells are being coated.
Sacrifice a wild type mouse and dissect the spleen, placing it into a 50 milliliter tube with media. Isolate the PLE cytes by placing the spleen between two microscope slides and mashing it over a Petri dish. Resus suspend the cells in PBS, pipette the cell suspension into a new 50 milliliter conical tube and centrifuge for five minutes.
At 400 Gs at room temperature, remove the snat and lyce the red blood cells by Resus suspending the cell pellet in four milliliters of act lysis buffer for two to three minutes. Stop the lysis with five milliliters of PBS centrifuge the cell suspension again for five minutes at 400 Gs at room temperature and discard the supernatant resus. Suspend the resulting pellet in one times 10 of the six cells per milliliter in complete ice.
Coves modified delcos medium or IMDM equally, divide the resulting cell suspension between two separate tubes, supplementing one of the tubes with all trans retinoic acid or RA to a final concentration of 100 to 200 nanomolar cubed. Protect the tube from direct light to avoid all trans retinoic acid degradation. Remove the 24 well plate from the incubator and wash each well twice with two milliliters of PBS.
Add 1.5 to two milliliters of the RA supplemented cell suspension to half of the 24 well plate. Then add the same amount of the control cells to each well of the second half of the 24 well plate and return the plate to the incubator. After two to three days, transfer the cell suspensions into a new uncoated 24 well plate.
To avoid overstimulation of the T cells, incubate the cells for an additional two to three days. If the media turns to yellow to the cell density and or proliferation, add 500 microliters of fresh IMDM to each well to reach a final volume of one milliliter per well. To harvest the cells, collect the supernatant on day four or five, counting the day the cells were plated as day zero.
Typical final yields are one to two times 10 of the six effector T cells per well. Therefore, a minimum of nine to 12 wells of each of the gut Tropic and controlled T cells should be plated in order to generate 10 to 20 times 10 to the six T cells per group. To check the levels of alpha four, beta seven, and CCR nine expression on ra.
Activated and controlled T cells collect between 0.2 and 0.5 times 10th of the sixth of the freshly harvested experimental and control cells into four milliliter fax tubes and centrifuge. The tubes for five minutes at 300 Gs at four degrees Celsius. Discard the supernatant and resuspend the cells in 100 microliters of staining buffer incubate with anti CD four FSE ANTIFA four beta seven pe, anti CCR nine a PC and anti CD eight per P for 15 minutes at four degrees Celsius in the dark.
After the incubation, wash the cells with PBS for five minutes at 300 GS four degrees Celsius, discard the supernatant and resuspend the cells in staining. Buffer again and analyze by flow cytometry at the beginning of this step. Prewarm a bottle of IMDM and a bottle of PBS at 37 degrees Celsius for later use in the staining protocol.
After confirming the T-cell homing phenotypes of the two cell cultures by flow cytometry as described in the previous step centrifuge, the freshly harvested alpha four, beta seven, high CCR nine, high gut tropic and alpha four, beta seven, low or negative CCR nine low or negative control T-cell populations at 300 Gs for five minutes at room temperature. Next, wash the cells two more times with PBS to remove the serum and then adjust the T-cell concentration for each group to 10 to 15 times 10 to the six cells per milliliter. In PBS add, CFSE or C-M-T-M-R dies to either gut tropic T or control T cells.
Here CFSE is being added to the control cell population and C-M-T-M-R is being added to the gut. Tropic T cells to a final concentration of five millimolar and 10 millimolar respectively gently vortex the cells for five seconds and incubate the tubes for 20 minutes at 37 degrees Celsius in the water bath after the incubation period. Halt the staining with the addition of five milliliters of FPS and incubate for one to five minutes at room temperature.
Dilute 10 times with the warm PBS and centrifuge for five minutes at 300 Gs at room temperature. Discard the supernatant and wash two more times with PBS and resuspend the cells in complete IMDM. Ideally, 10 to 20 times 10th of the six cells of each T-cell population should be mixed together in four milliliter fax tubes at a one-to-one ratio.
To calculate the exact input ratio, save five to 10 microliters of the tubi injected cell suspension, which is called input and keep it at four degrees Celsius centrifuge the cell mixture to be injected at 300 Gs for five minutes. At room temperature. Re suspend the cells to be injected in 250 to 300 microliters of the warm PBS load, A one CC insulin syringe with a 28 gauge needle with the cells for injection.
Inject 250 microliters of the cell mixture via the tail vein. Finally, analyze the input cells by flow cytometry to obtain the percentage of CFSE and C-M-T-M-R positive cells that were previously injected. That will be the input ratio used.
In the final calculation of the homing index. We suspend the cell samples from the tissues for analysis of T-cell homing at a density no higher than three times 10 of the six cells per milliliter. If congenic mice are used as recipients for this video, CD 45.1 positive were used stained with the appropriate congenic marker combined with some lineage marker during flow cytometric analysis gate the cells on the chosen congenic marker and then on the specific lineage marker CD eight, and analyze the ratios between C-M-T-M-R and CFSE positive cells.
The data is then expressed as the homing index or hi, which is calculated as the ratio between the two different dyes in each tissue divided by the corresponding input ratio. The input ratio is the ratio of the CFSE positive to CMTM TMR positive cells in the syringe prior to injection calculated from the five to 10 microliter aliquot of cell suspension that was set aside and analyzed by flow cytometry for this purpose. If the input ratio is very close to one, then the tissue ratios will be equivalent to the hi.
If the HI and the blood is significantly different than one, the HI in the tissues being analyzed can be normalized by the HI in blood by calculating the ratio of the HI in the tissues divided by the HI in the cell suspensions generated from the spleen, peripheral lymph nodes or PLN mesenteric lymph nodes or MLN and small intestine Lamin propria or LP 18 hours after injection. Were analyzed for C-M-T-M-R and CFSE expression on gated CD eight positive CD 45.1 positive cells RA activated alpha four, beta seven and CCR nine expressing C-M-T-M-R positive T-cells home to the spleen in equal numbers to control CFSE positive cells, but exhibited a marked increase in homing to the gut and training lymph tissues calculation of the HI for each tissue. Further confirmed that the gut tropic and control T cells homed equally to the spleen, but by contrast, the gut tropic T cells migrated approximately 10 times more efficiently to the LP as compared to control T cells After mastering the procedure.
Using congen mice instead of dye leveling can be done in order to assess the migratory ability of additional cell types such as dendritic cells or microphages. Since these cell types tend to lose the dye once injected into the recipient mice, using congenic animals allows for better tracking of the cells and for much longer time periods after adopted transfer.
