Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

1. Preparation

1. Before beginning, put on laboratory gloves and appropriate protective clothing.
2. Wash all the dissection tools, first with a detergent and then with 70% ethanol and then dry them with a clean paper towel.
3. Prepare 200 mL of Hank's balanced salt solution (HBSS) containing 2% fetal calf serum (FCS).

2. Dissection

1. Pin a euthanized mouse on a dissection plate in the supine position.
2. Using scissors and forceps perform a longitudinal laparotomy to access the chest cavity.
3. Remove the heart to gain access to the thymus, which is located above the heart. Then, identify the thymus, which is composed of two white lobes and is located in the chest cavity above the heart.
4. Using forceps carefully detach the thymus and place it on the Petri dish with 5 mL of HBSS 2% FCS.

3. Immune cell isolation

1. Place the thymus on a 40 µm cell strainer over the same Petri dish. Crush the thymus with a plunger to dissociate it in the same dish.
2. Transfer the dissociated thymus and the fluid into a 15 mL centrifuge tube.
3. Wash the Petri dish with 5 mL of HBSS 2% FCS and transfer the washed medium also into the same centrifuge tube.
4. Centrifuge the tube at 370 x g for 7 min at 20°C and discard the supernatant avoiding the pellet.
5. Resuspend the pellet in 2 mL of potassium acetate to lyse the erythrocytes. Wait for 2 min and then make up the volume up to 14 mL using HBSS 2% FCS.
6. Centrifuge the tube again at 370 x g for 7 min at 20°C. Discard the supernatant and resuspend the pellet in 5 mL of HBSS 2% FCS.
7. Estimate the cell concentration using the trypan blue staining assay and adjust the final cell concentration to 10⁷ cells/mL using appropriate volume of HBSS 2% FCS.

4. Magnetic labeling of immune cells

1. Take two FACS tubes. Label one tube, "non-enriched T cells", and the other tube, "enriched T cells"- which will be separated using magnetic labeling.
2. Distribute of cell solution into each of the two FACS tubes.
3. Centrifuge the "enriched T cells" tube at 370 x g for 3 min at 20°C and discard the supernatant avoiding the pellet.
4. Resuspend the pellet in 250 µL of anti-CD3 biotinylated antibodies mix (Table 1, Mix 1).

Mix	Labeling reagents	Dilution
1	Anti-CD3 biotinylated antibody	1/400 (in HBSS 2% FCS)
2	Streptavidin coupled beads	1/5 (in HBSS 2% FCS)
3	Anti-CD3 BV421	1/200 (in HBSS 2% FCS)

Table 1: Antibodies mix composition. Mixes 1 and 2 are used for magnetic separation. Mix 3 is used for evaluating the cell enrichment after magnetic separation.

5. Incubate the cell suspension-antibody mix for 15 minutes at 4°C in the dark.
6. Add 3 mL of HBSS 2% FCS to both the tubes and centrifuge them again at 370 x g for 3 min at 20°C.
7. Discard the supernatant and resuspend the pellet in 250 µL streptavidin-coupled beads (Table 1, Mix 2).
8. Incubate the cell mixture and beads for 20 min on ice.
9. Next, add 3 mL of HBSS 2% FCS and mix well and centrifuge again at 370 x g for 3 min at 20°C.
10. Resuspend the pellet in 2 mL of HBSS 2% FCS.

5. Magnetic Separation of CD3-Positive Cells

1. Place the column on the magnet and add 3 mL of HBSS 2% FCS to humidify the system. Wait 5 minutes.
2. Next, pipet the labelled cells into the column.
3. After the cell suspension passes through the column, wash the column X3 times with 3 mL of HBSS 2% FCS.
4. Then, remove the column from the magnet and place it in a 15 mL collection tube.
5. To elute the target cells, add 5 mL of HBSS 2% FCS to the column and flush the column with the plunger.
6. Repeat elution step with another 5 mL of HBSS 2% FCS.

6. Evaluation of Target-Cell Enrichment by Flow Cytometry

1. Transfer 500 µL of eluted cell suspension to a FACS tube labeled "enriched T cells". Transfer 200 µL of "non-enriched T cells" suspension to a second FACS.
2. Then, centrifuge both the tubes at 370 x g for 7 min at 20°C.
3. Discard the supernatant, then add 100 µL of fluorescent antibody Mix 3 (see Table 1) to both tubes.
4. Incubate both the tubes for 20 min at 4°C in the dark.
5. Next, add 3 mL of HBSS 2% FCS to tubes and centrifuge them at 370 x g for 3 min at 20°C.
6. Discard the supernatant, then resuspend each tube in 250 µL of HBSS 2% FCS.
7. Now, evaluate the CD3-positive cell enrichment rate by flow cytometry.

7. Data Analysis

1. Open the 'FlowJo' icon and drag the files for each tube in the "All Sample" window.
2. Double click on the "enriched T cells" file to display the dot plot displaying forward scatter (FSC-A) on the X-axis and side scatter (SSC-A) on the Y-axis.
3. Click on "Polygon" to circle the lymphocyte populations.
4. Next, double click on the circled population to create a new window.
5. Select "FSC-W" on the Y axis and "FSC-A" on the X axis, and circle the FSA-W negative cells. In the "Subpopulation identification" window, name the cell population "single cells".
6. Double click on the circled population to create a new window. Select "CD3" on the Y axis, and circle the CD3-positive cells. In the "Subpopulation identification" window name your cell population "T cells".
7. Repeat with the "non-enriched T cells".
8. To visualize cell population, click on "Layout editor" and drag the "T cells" population from "enriched T cells" and "non-enriched T cells" files into the tab.
9. Dot plots representing CD3+ lymphocytes will appear. CD3+ cells should only appear in the population of interest in the CD3+ enriched tube.
10. To evaluate the enrichment of CD3+ lymphocytes in the sorted cells, click on "Table editor", and then drag the "T cells" population from "enriched T cells" and "non-enriched T cells" files into the table.
11. On the "Statistic" menu, select "Frequency of lymphocytes" cells to check the percentage of CD3+ cells in all lymphocytes, then click on "Create table".
12. Parameter values will appear in a new table. For the "enriched T cells", the frequency of CD3+ cells should be around 80%.