The overall goal of the following experiment is to assess the functional quality of CD eight positive T cells in response to a particular antigen by investigating their ability to kill target cells in vivo. This is achieved by first eliciting antigen specific effector CD eight positive T cells by immunizing recipient mice. As a second step, primary target cells are given specificity or are otherwise treated, which completes the creation of the specific effector target system.
Next, the target cells are given to the recipient mice via retroorbital adoptive transfer. In order to look at the effector killing in vivo results were obtained that show whether the effectors have killed any of the targets based on flow cytometric analysis of CF SE stain cells. Hi, my name is Marina Ward.
I'm a graduate student in the lab of Gary Splitter at the University of Wisconsin Madison. This method will provide insight into CTL killing and can also be applied to cell proliferation and survival. Demonstrating the adoptive transfer procedure will be Dr.Jerome harms a scientist from my laboratory Start strategically by defining a specific effector target system.
For example, in a classical in vivo CTL assay, use a purified peptide immunization to elicit specific CD eight positive T cells use the same peptide to pulse the syngeneic target cells, thus creating a specific effect to target system. Decontaminate the work surface of a biosafety hood to ensure sterility of the injections. Prepare the peptide immunization by first placing into a two milliliter tube.
50 micrograms of purified peptide into 100 microliters of 10%DMSO in PBS per mouse. Next, add an equal amount of very cold, incomplete free unju with one millimeter beads for mixing. Secure the tube lid with paraform and store the immunogen on ice until injection.
Use a bead beter set to maximum rotations per minute to mix the emulsion for three minutes promptly placing the emulsion back on ice after mixing. The resulting emulsion should be similar in consistency to mayonnaise. Allow the emulsion to sit at four degrees Celsius overnight or up to a week mixing further if any separation is noted at the time of immunization.
Identify the mice for this part of the experiment. Use a one milliliter syringe and a 20 gauge needle to pull up the emulsion. Being careful not to pull up any beads in order to prepare target cells.
Naive syngeneic mice are used euthanize these naive mice. According to A-A-A-L-A-C approved standards. Spray the relevant area of the mouse with 70%ethanol to minimize the chance of contamination from mouse, hair or other external contaminants using dissection scissors, make a small snip through the skin on the left side of the mouse.
Pull the skin flap over to uncover the peritoneal sack and internal organs. Cut through the peritoneum being careful not to compromise any of the underlying organs. Locate the spleen which is close to the surface and dark red in color.
Carefully pull out the spleen and immediately place it into a 15 milliliter conical tube containing 10 milliliters of prepared RPMI medium on ice. Continue with the remaining mice. Transfer the spleen and medium into a glass tissue.Homogenizer.
Grind the spleen until all tissue has lost its color. Pass homogenized products through a 70 micron cell filter into a 50 milliliter conical tube. Rinse the homogenizer once with fresh medium and combine with the spleen homogenate by pouring through the filter centrifuge samples at four degrees Celsius and 1, 300 rotations per minute.
For seven minutes, decant and discard supinate. Re suspend the cell pellet in 10 milliliters of lies, reagents and incubate at 37 degrees Celsius for seven minutes. Add up to 50 milliliters with fresh, medium, and harvest cells by centrifugation.
Repeat this wash. Count the cells and aliquot 10 million cells per well. In a 96 well round bottom tissue culture plate.
Incubate at four degrees Celsius for one and a half hours. Move to the 37 degree Celsius incubator for the last 30 minutes. Prepare fresh CFSE reagent according to manufacturer and dilute to a work in concentration of between 0.5 and 25.Micromolar.
Make two CFSE solutions that differ tenfold in concentration. Equilibrate these low and high concentration CFSE solutions to 37 degrees Celsius before labeling. Remove the 96 well plate of target cells from the incubator and pellet cells by centrifugation.
At room temperature, flick the plate to remove SUP natant. Resuspend the cells in 200 microliters of either low or high concentration CFSE solution and incubate for 15 minutes in a 37 degree Celsius incubator pellet cells by centrifugation at room temperature, flick the plate to remove SUP natant Reeses bend the cells in 200 microliters of fresh, medium and incubate for 30 minutes. At 37 degrees Celsius pellet cells again by centrifugation, discard, supinate and resuspend the pellets in 50 microliters of PBS set aside some of the labeled cells for non-transfer controls during data analysis and also for running on the flow cytometer for the adoptive transfer.
Add one well of low concentration CFSE labeled cells to one well of high concentration CFSE labeled cells per mouse. Thus, the resulting injection consists of 50%specific targets and 50%irrelevant cells in 0.1 milliliter. In order to facilitate the adoptive transfer anesthetize the peptide immunized mice inject 0.1 milliliter of labeled target cells by a retroorbital route into the recipient.
Mice after six hours, euthanize the recipient mice for cell harvest, isolate the mouse spleen and purify the cell population by the same protocol that was used. When preparing target spleen cells, plate cells at 1 million per well into a 96 well round bottom tissue culture plate, fix the recovered and non transferred cells with para formaldehyde, run samples on a flow cytometer and analyze the data. Typical presentation of results of cell specific killing is depicted in two ways.
A diminished epitope specific peak on the fax histogram and the calculated value is cell specific lysis. Here the numbers indicate the percentage of cells of the high or low CFSE phenotype in non transferred control CFSE labeled cells recovered from A PBS immunized recipient mouse and CFSE labeled cells recovered from two different peptides specific recipient mice. Note the diminished epitope specific peaks indicative of cell specific killing.
Alternatively, the results can be presented graphically to indicate cell specific lysis for each group of mice. PBS, peptide A and peptide B.After watching this video, you should have a good understanding of how to develop your own in vivo CTL killing assay using mice.
