The overall goal of the following experiment is to examine thy development in mice by flow cytometry. This is achieved by preparing single cell suspensions from the mouse, thymus and spleen. As a second step, the thymocytes and cytes are stained with a cocktail of antibodies for the identification of specific thy populations.
Ultimately, the frequency and number of various lymphocyte populations can be evaluated by flow cytometric analysis. This method can help answer key questions in T cell development, such as which genes and pathways are important for generating a functional yet cell tolerant T cell repertoire. Though this method can provide insight into T-cell development in the thymus, it can also be applied in the examination of the development of other immune cells.
Generally, individuals new to this method will struggle in the design of their antibody cocktails for flow cytometry. Before starting the dissection, place a sterile steel mesh screen into a 60 by 15 millimeter Petri dish. Then add five milliliters of HBSS to the dish and store the dish on ice to harvest the thymus.
First, use a pair of forceps to lift the bottom tip of the sternum and reveal the diaphragm. Next, avoiding the liver, cut the diaphragm to detach the ribcage and then cut the ribcage in an upwards direction on each side. Taking care to avoid the lungs and heart.
Now use the forceps to gently pull the rib cage back. The thymus is a white bi lobed organ located above the heart. Use the flat edge of the forceps to grasp the bottom of the lobes.
Then gently pull the thymus out and place it onto one of the previously prepared mesh screens. To harvest the spleen, make an incision through the peritoneum to expose the abdominal cavity. The spleen is a red surfboard shaped organ located on the left side of the mouse's abdominal cavity below the liver.
Then use one pair of forceps to pull out the spleen and another to tease away the connective tissue. Then place the dissected spleen on a separate mesh screen. Now use the plunger from a three milliliter syringe to grind each organ into its mesh screen until only connective tissue and fat remain pipette the cells and HBSS into a 15 milliliter conical tube.
Then rinse each mesh with fresh HBS S3 times. Next, pellet the cells for five minutes at 335 times G and four degrees Celsius. After aspirating the supernatant resus, suspend the cytes in 500 microliters of a CK lysis buffer for 10 minutes at room temperature while the splenic red blood cells are being lyed Resus.
Suspend the thymocytes at 20 times 10 to the six cells per milliliter in fax buffer, and set them aside on ice. Now add five milliliters of HBSS to return the cytes to isotonicity after spinning down the cells again, resus suspend the RBC free pellet at 20 times 10 to the sixth cells per milliliter in fax buffer. Begin this step by Ali Watting.
Four times 10 to the sixth of the reserved THYMOCYTES per flow cytometry sample to each well of a 96 well plate. Next, add cytes to the wells of a 96 well plate for compensation controls. Then block the FC receptors in each of the cell samples by incubation with anti CD 1632 for 10 minutes on ice.
Now spin down the plate and then dispel the liquid from the wells by flicking the plate once face down into a sink, then resus suspend each well in 200 microliters of fax buffer and repeat the wash. Next, incubate the experimental samples with 200 microliters of antibody cocktail or single fluorochrome conjugated antibodies for the compensation controls. All in fax buffer on ice in the dark.
After 30 minutes, wash the cells twice with fax buffer and then after resus suspending the cells in fax buffer transfer the samples to fax tubes in physiological TCR transgenic models and wild type mice. Positive selection begins at the double positive bright stage before moving into the double positive dull stage. After antigen encounter the double positive dull thymocytes, then enter a transitional CD four positive CD eight low stage before becoming CD four.
Single positive or CD eight single positive thymocytes mature single positive thymocytes are characterized by their high TCR expression and loss of CD 24. While the CD eight by CD four profile can reveal defects in positive selection, examining TCR beta by CD 69 or CD five can provide further insight into where the defect lies. Both CD 69 and CD five are upregulated after TCR stimulation with stronger interactions driving higher expression of these markers.
In this TCR beta by CD 69 plot in a wild type mouse, the TCR R beta low CD 69 negative gait represents a population of pre-selection double positive thymocytes. Whereas this TCR beta intermediate CD 69 positive gait represents a transitional population directly after TCR engagement and consists of double positive bright with some double positive dull and CD four positive CD eight low cells. Here the TCR R beta high CD 69 positive gait illustrates the population of cells directly post positive selection, which consist of double positive dull CD four positive, CD eight low and CD four single positive cells.
Finally, this TCR beta high CD 69 negative gait shows a more mature population of cells that primarily consists of CD four and CD eight single positive cells. The absence of the TCR R beta intermediate CD 69 positive and TCR R beta high CD 69 positive populations may be indicative of impaired positive selection changes in the ratio of the CD four single positive to CD eight. Single positive cells within the TCR beta high CD 69 negative population may suggest alterations in lineage commitment.
The loss of the TCR beta high CD 69 positive and TCR beta high CD 69 negative populations may reflect survival issues following positive selection. Examining TCR beta by CD five is another strategy to identify pre and post positive selection populations. These first two populations consist primarily of double positive bright thymocytes.
The TCR beta low CD five low population represents preselection double positive thymocytes, and the TCR beta intermediate CD five intermediate population is made up of the cells that initiate positive selection. Impaired generation of this or the previous population suggests defective positive selection. The TCR R beta intermediate CD five high population, however, represents thymocytes in the process of undergoing positive selection and consists primarily of double positive dull and CD four positive.
CD eight low thymocytes. The TCR beta high CD five high population consists primarily of post positive selection. Single positive thymocytes changes in the ratio of CD four, single positive to CD eight.
Single positive cells within this population, despite normal preceding populations may suggest alterations in lineage commitment. Further, an absence of this population may indicate a decreased survival of thymocytes following positive selection as negative selection involves the deletion of small antigen specific populations. Defects in this process are best observed using TCR transgenic mice in HY CD four mice thymocytes expressing the transgenic H-Y-T-C-R can be detected with the monoclonal antibody T 3.7.
The H-Y-T-C-R recognizes the male specific HY antigen presented within MHC Class one db. Thus HY CD four male mice undergo negative selection of H-Y-T-C-R positive thymocytes as indicated by a reduction in T three point 70 positive double positive THYMOCYTES numbers, and a more dramatic decrease in T 3.7 positive CD eight, single positive thy cyte numbers. In contrast, HY CD four female mice undergo positive selection to generate T 3.7 positive CD eight single positive T cells.
While a reduction in double positive thy numbers is indicative of negative selection, the absence of antigen specific single positive thymocytes is the most accurate measure. Further examination of the few T three point 70 positive CD eight single positive thymocytes in HY CD four male mice reveals that most are CD 24 high immature cells. While most T three point 70 positive CD eight single positive thymocytes in the HY CD four female have reached maturity and are CD 24 low, providing further support, that negative selection occurs in HY CD four male mice.
One caveat of the TCR transgenic models is that it is difficult to further characterize positive selection by identifying populations based on TCR and CD 69 or CD five expression due to high TCR expression throughout thy CYTE development, HY CD four female mice have a large population of T three point 70 positive double positive thymocytes that undergo positive selection. As indicated by an increase in the CD 69 positive population compared to wild type negative selection involves a higher affinity TCR stimulus than positive selection. This is indicated by higher expression of CD 69 on T three point 70 positive double positive thymocytes in HY CD four male mice resulting in a shift of the histogram peak to the right.
Similar trends are seen with CD five expression when analyzing thymic selection in genetically manipulated mice. Examining CD 69 or CD five expression can determine whether the defect lies with the TCR signaling or a downstream outcome of TCR stimulation. Once mastered, this technique can be performed in two and a half hours for cell preparation and staining, plus an additional one hour for data collection on the flow cytometer.
If performed correctly, each additional mouse will add approximately 30 minutes. While attempting this procedure, it is important to remember to handle the thymocytes gently and to ensure you have planned the staining strategy beforehand. Following this procedure.
Other assays such as killing assays or cytokine production assays can be performed in order to answer additional questions like whether the exported thymocytes function properly. After watching this video, you should have a good understanding of how to analyze thymic site development using flow cytometry.
