The overall goal of this procedure is to differentiate TH 17 cells from naive CD four positive T lymphocytes. This is accomplished by first harvesting the lymph nodes and spleen from an adult C 57 black six mouse. In the second step, the tissues are ground to obtain a single cell suspension.
The CD four positive CD 25 negative T cells are then isolated and cultured under TH 17 inducing or activation control conditions. Ultimately Q-P-C-R-E-I-S-A and flow cytometry can be used to evaluate IL 17 A expression. So this method can provide insight into the differentiation of CD four T lymphocytes into T eight 17 cells in C 57 black six mice.
It can also be applied to other mouse models as well. Demonstration of this method is critical as the lymph nodes are very small, making the dissection steps difficult. Without direct visualization of this method, At least four hours before adding the cells coat the wells of a new 96 well UBO micro test tissue culture plate with 30 microliters of anti CD three diluted in sterile PBS tap the sides of the plate to ensure uniform coverage of the wells and then incubate the plate at 37 degrees Celsius after four hours, refrigerate the plate until it is time to add the cells.
After confirming death by cervical dislocation, sterilize the area of incision on an adult C 57 black six mouse with 70%ethanol. Then grab the skin anterior to the urethral opening and cut from the ventral midline to the chin area, taking care not to disturb the peritoneum. Next pin down the skin to allow access to the lymph nodes and spleen for removal.
Now remove the axillary lymph nodes found near the axilla of the mouse behind the pectoral muscles and place the tissue in a Petri dish containing five milliliters of AutoMax running buffer. Then remove the brachial lymph nodes located in the connective tissue near each axilla. Next, remove the superficial cervical lymph nodes found in the animal's neck, flanking the trachea, and then remove the inguinal lymph nodes located in the hip region at the conjunction of the three blood vessels.
To access the mesenteric lymph nodes first cut up the ventral midline through the peritoneal lining. The mesenteric lymph nodes are located deep within the mesentery of the mouse, generally in a string of four to eight nodes that may appear as a string of pearls. Remove the spleen pulling and detaching it from the pancreas.
Then place it in the Petri dish with the lymph nodes to grind the organs. Place the lymph nodes on the frosted side of one microscope slide and then rub the tissues with the frosted side of a second slide. To filter the ground organs into a single cell suspension, first fold a roughly three inch by three inch piece of 40 micron nylon material several times and place it in the top of a 15 milliliter centrifuge tube.
Use a five milliliter syringe equipped with a 21 gauge needle to aspirate the cells and buffer and then dispense the cell solution slowly into the folded nylon. Taking care to avoid puncturing the material with the needle. Once a single cell suspension has been achieved, the solution should appear consistent with no visible pieces of solid tissue.
Place this solution on ice. After sorting the cells by AutoMax separation to an at least 80%CD four positive CD 25 negative cell purity. Count the cells by trian blue exclusion and then dilute the cell suspension to one times 10 to the six cells per milliliter in cell culture media.
Now rinse all the wells of the anti CD three coated plate with 200 microliters of PBS. Each discarding the wash into a waste container. Then add 100 microliters of the TH 17 inducing cocktail or activation control mixes to the appropriate wells in triplicate.
Finally, add 100 microliters of cells to each well and incubate the cells for at least 72 hours or up to five days to achieve TH 17 cell differentiation for Eli SA and QPCR. After the incubation period, pool triplicates of each sample into individual einor tubes. After spinning down the cells, transfer the supernatant into separate einor tubes and store at minus 20 degrees Celsius to later measure IL 17 A production by ELI.
A.To prepare for QPCR after removing the supernatant resuspend, the remaining pellets in 175 microliters, RNA lysis buffer for immediate RNA extraction or store at minus 80 degrees Celsius until ready to begin the procedure after the incubation period. In preparation for cell activation prior to IL 17, a intracellular staining pool each of the TH 17 inducing or activation control cell triplicates into individual wells of a 24 well cell culture plate. Add 400 microliters of cell culture medium to each well.
To bring the total volume up to one milliliter, then add PMA ion mycin and felden A to each well and incubate the cells for four hours at 37 degrees Celsius to detect the presence of IL 17 A by intracellular staining and flow cytometry. First, transfer the cells from each well of the 24 well plate into a separate einor tube. Then spin down the tubes.
Remove the supernatants and resuspend the cell pellets in 200 microliters of fax buffer. Transfer the resuspended cells to a 96 well flow cytometry plate and spin down the cells again after washing the pellets in 200 microliters of fax buffer resuspend the cells in 100 microliters of fax buffer and add 100 microliters of the extracellular antibody staining mixture. Incubate for 15 minutes at room temperature away from light.
After washing the cells twice, incubate the pellets in 100 microliters of BD cyto. Fix cyto perm buffer covered in foil for 20 minutes at room temperature. Now wash the cells twice in 100 microliters of one X BD perm wash buffer, and then incubate the cells in 50 microliters of intracellular antibody mixture covered in foil for 15 minutes at room temperature.
After washing the cells again resus, suspend the cells in 200 microliters of BD staining buffer, and then transfer the cells into flow cytometry tubes containing 200 microliters of the staining buffer. Store the tubes at four degrees Celsius until they are ready to be read for flow cytometric analysis of the samples first gate on the live cell population. Next, use the live cell population to gate the CD four positive CD eight negative population.
Finally from the CD four positive CD eight negative population gait on the IL 17 A positive population to differentiate TH 17 cells, the unfractionated pooled lymph node and spleen cells must be enriched for the CD four positive CD 25 negative T-cell population. In this figure, unfractionated pooled lymph node cells, SP cytes and AutoMax separated TT cell fractions stained with anti CD four and anti CD 25 are shown. This first scatter plot shows a representative profile of the percentage of CD four positive, CD 25 positive and CD four positive CD 25 negative lymphocytes present in the unfractionated pooled lymph nodes and spleen cells.
The isolation procedure also provides an enriched CD four positive CD 25 positive treg population that can be used in additional experiments. In this final scatter plot, the desired cell enrichment with a population that is 84%CD four positive CD 25 negative T-cell is shown. This enriched CD four positive CD 25 negative T-cell population helps to ensure a more successful TH 17 differentiation as illustrated in this figure.
Whereas incubation of CD four positive CD 25 negative T lymphocytes with anti CD three and anti CD 28 for five days yield CD 25 positive T lymphocytes incubation under TH 17 inducing conditions yields a subset of IL 17 producing CD four positive CD 25 positive lymphocytes. TH 17 differentiation status can be further assessed through quantitative PCR and Elis A here data from enriched CD four positive CD 25 negative T lymphocytes incubated under control or TH 17 inducing conditions is shown CD four positive. CD 25 negative T lymphocytes incubated under TH 17 conditions upregulate IL 17 A, which can be ascertained through QPCR and ELI SA.The TH 17 specific transcription factor ROAR gamma T is also upregulated by CD four positive CD 25 negative T cells incubated under TH 17 inducing conditions as can be measured by QPCR.
While attending this procedure, it is important to remember to achieve at least an 80%CD four positive CD 25 negative cell purity before incubating the cells under TH 17 inducing conditions. After watching this video, you should have a good understanding of how to dissect lymph node and spleen from C 57 black six mice, how to incubate cells under T eight 17 or activation control conditions, and how to assess the differentiation of naive CD four T lymphocytes into T eight 17 cells South.
