Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

This protocol helps in investigating the role of immune cells in particular organs in health and pathology. The main advantage of this technique is that this is a cost-effective technique since it's a manual technique. Demonstrating the procedure will be Zhengkang Luo, a PhD student from my laboratory.

To begin, place the thymic glands and spleens from previously euthanized mice into 20 ml scintillation phials, or 15 ml conical tubes, filled with 5 ml of Hank's balanced salt solution. Place the pancreatic draining lymph nodes into 1.5 ml micro-tubes filled with 1 ml of RPMI-1640. Make sure to use the whole thymus and spleen, and all of the PDLNs.

Keep the organs on ice throughout the entire procedure. Using a pair of scissors, thoroughly squeeze the thymus and spleen to release the immune cells. Discard the remaining thymic and splenic capsules.

Transfer the cell suspension to a 15 ml conical tube. Centrifuge at 433 times g and at 4 degrees celsius for five minutes, and discard the supernatant. To lyse the red blood cells, re-suspend the cell suspension in 5 ml of 0.2 Molar ammonium chloride, and incubate at room temperature for ten minutes.

Invert the tubes gently every two minutes. At the end of the incubation, add 5 ml of HBSS to stop the lysis. Centrifuge the tubes at 433 times g and at 4 degrees celsius for five minutes.

Discard the supernatant, and re-suspend the cells in approximately 5 ml of HBSS. Then, fill the tubes with HBSS. Repeat this process, from centrifuging the samples to filling the tube with HBSS, one time.

Centrifuge the tubes once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in HBSS. Obtain 5 ml round-bottom tubes with cell-strainer caps.

Transfer 1 ml of the thymic cell suspension and 500 microlitres of the splenic cell suspension to the tubes by applying the suspension to the cell-strainer caps. First, place a 15 ml conical tube into a rack. Place a sterile 250 micrometer metal mesh over the tube.

Rinse the mesh with 1 ml of RPMI. Next, transfer the lymph nodes to the metal mesh and use a pair of tweezers to grind them through the mesh. Apply 1 ml of RPMI on the mesh to flush the cells into the tube.

Repeat this process of transferring and grinding the lymph nodes three times for each sample, and then remove the mesh. Centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cells in approximately 5 ml of RPMI.

Then, fill the tubes with RPMI. Repeat this process, from centrifuging the samples to filling the tube with RPMI, one time. Centrifuge the tubes again at 433 times g and at four degrees celsius for five minutes.

Discard the supernatant and re-suspend the cell pellet in 2 ml of RPMI. Transfer 2 ml of the cell suspension into 5 ml round-bottom tubes with cell-strainer caps. First, centrifuge the cell suspension from the thymus, spleen and PDLN samples at 433 times g and at four degrees celsius for five minutes.

Discard the supernatant. Stain the cells with surface antibodies as outlined in the text protocol, and incubate the tubes on ice for 40 minutes. Next, add 200 microliters of FACS buffer to each tube.

Centrifuge at 433 times g and at four degrees celsius for five minutes, and discard the supernatant. Repeat this process, adding the buffer, centrifuging, and discarding the supernatant, one time. Re-suspend the cell pellet in 500 microliters of permeabilization fixation buffer to fix and permeabilize the cells.

Transfer the tubes to a refrigerator at four degrees celsius overnight. The next day, centrifuge the tubes at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the cell pellet in 500 microliters of permeabilization washing buffer.

Centrifuge the cells again at 433 times g and at four degrees celsius for five minutes and discard the supernatant. Stain the cells with intracellular antibodies, as outlined in the text protocol. Incubate the tubes on ice for 1 hour.

After this, add 500 microliters of permeabilization washing buffer to each tube. Centrifuge at 433 times g and at four degrees celsius for five minutes. Discard the supernatant, and re-suspend the pellet in 500 microliters of permeabilization washing buffer.

Centrifuge once more at 433 times g and at four degrees celsius for five minutes. Discard the supernatant and re-suspend the cell pellet in 300 microliters of FACS buffer. Then, analyze the cells on a flow cytometer, as outlined in the text protocol.

In this study, single cells are isolated from thymic glands, PDLNs and spleens of normal glycemic NOD mice, and are stained with Treg cell markers, CD4, CD25, Foxp3, Helios, and neuropilin-1 for flow cytometric analysis. The results are analyzed and are shown here as representative gating strategies. The proportion of Helios-positive cells among the CD4-positive, CD8-negative, CD25-positive, Foxp3-positive Treg cells is seen to be higher than that of Nrp1-positive cells in all three organs.

More than 80%of Treg cells in the thymus are seen to express Helios, which is higher than in the PDLN, and the spleen. The proportion of NrP1-positive cells among Helios-positive Treg cells is seen to be higher in the PDLN than those in either the thymus or spleen. The majority of Nrp1-positive Treg cells also express Helios, and the proportion of Helios-positive cells among Nrp1 positive Treg cells is seen to be higher in the thymus and spleen than in the PDLN.

Together, these results indicate that Helios is a better marker to detect T Treg cells than Nrp1. Lymph nodes are small, but some intracellular markers require a large number of cells to give a good signal in flow cytometry. Other methods cannot be performed after this procedure because the cells are already stained and dead.

But the animal's four cell markers can be replaced to study other types of immune cells.