The overall goal of the following procedure is to polarize naive CD four positive T cells isolated from mouse lymph nodes, and spleens into distinct T helper subsets. In vitro, this is accomplished by first processing spleens and lymph nodes harvested from adult wild type mice. In the second step, the CD four positive cell population is enriched by magnetic bead separation and then further sorted by their naive T-cell marker expression.
In the final step, the cells are treated with T-cell receptor activating factors and polarizing cytokines to induce their differentiation into various T helper lineages. Ultimately flow cytometry. Elis A and real-time PCR are used to assess the efficiency of the T helper cell differentiation.
This method can help answer key questions in the T helper cell field, such as how does the expression of specific molecules or the treatment with specific drugs influence the nature of a T cell response. Begin by pinning the limbs of a five to 10 week old C 57 black six mouse to the dissecting board, and then cut the skin longitudinally from the anus to the chin, taking care not to puncture the underlying peritoneal tissue. Next, pin the skin open and use one pair of forceps to grab the lymph node and another to separate away the tissue from each of the locations indicated in the image.
As each lymph node is harvested, place it into a collection dish containing PBS supplemented with 1%FBS or PBS plus, and then carefully open the peritoneum and remove the spleen. Place the spleen into the collection dish as well, and transfer the dish to an ice bucket to enrich for the CD four positive cells. Split the lymph nodes in the spleens into two separate 60 millimeter dishes containing three milliliters of PBS plus each using sterile forceps.
Next place a square of sterile nylon mesh over the spleens and use the thumb side of a syringe plunger to gently smash them against the mesh until almost all of the tissue goes into suspension. Pipee the suspension up and down a few times to break up the remaining soluble clumps and then place a new piece of nylon mesh over the opening of a 15 milliliter conical tube. Filter the suspension into the tube to remove any remaining debris.
Now repeat the mechanical dissociation for the lymph nodes and fill both tubes of filtered cells with PBS plus. Then invert the tubes a few times and spin down the cells. Re suspend the lymph node pellet in two milliliters of PBS.
Add one milliliter of ice cold, a CK solution per mouse to the spleen cell pellet. After one minute, stop the red blood cell lysis with 10 milliliters of PBS plus. Invert the tube and spin down the cells again.
Then resuspend the spleen pellet in 10 more milliliters of PBS plus, add the lymph node cells and spin down the pooled cell suspension. This time resuspend the pellet in 15 microliters of CD four magnetic beads diluted in 85 microliters of PBS plus per mouse for 15 to 30 minutes at four degrees Celsius. Then dilute the cells in 10 milliliters of PBS plus.
Filter the suspension through a nylon strainer into a new 15 milliliter tube and spin down the cells. Again, re spin the pellet in 100 microliters of PBS plus per mouse and positively select for the CD four positive cells by magnetic bead selection according to the manufacturer's instructions. After obtaining the CD four positive, positive cell fraction, wash the cells in 10 milliliters of PBS plus to sort out the CD four positive T-cell fraction.
Next, label the cells with the appropriate fluorescent antibodies for 15 to 30 minutes at four degrees Celsius in the dark after washing. Filter the labeled cells to remove any debris and transfer them into a fax tube on ice and protect it from light sort the CD four positive, CD 25 negative CD 62 L, positive CD 44 negative population into complete RPMI medium. When all of the cells have been collected.
Wash the naive T cells in fresh, complete medium. Then resuspend the pellet and complete RPMI count the naive T cells and adjust the concentration to one times 10 to the six cells per milliliter to induce in vitro T helper subset differentiation. Next, wash each well of cell culture plates previously coated with anti CD three and anti CD 28 in one milliliter of sterile FBS free PBS and plate.
The cells finally supplement the culture media with the appropriate cytokines and blocking antibodies as outlined in the table and incubate the cells at 37 degrees Celsius with 5%carbon dioxide for four to five days after four to five days in culture. Intracellular cytokine staining of the CD four positive T cells from the in vitro differentiated TH one, TH two inducible treg and TH 17 cultures can be performed. To confirm that each T cell subset exhibits the expected cytokine profile.
Indeed, after 24 hour restimulation with anti CD three EISs, A shows that the subsets exhibit the corresponding extracellular production of the intracellularly expressed cytokine proteins as well. Real-time PCR for lineage specific transcription factors further verifies the appropriate transcription factor profiles of the in vitro differentiated CD four positive T-cell subsets. After watching this video, you should have a good understanding of how to isolate and process mirroring lymph node and spleen cells to obtain naive CD four positive T helper cells.
Furthermore, you'll be able to differentiate these T cells into TH one, TH two TH 17, or ireg cells.
