Immunological Isolation of H2DM Compartments From TEC Lines

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3.4.1. Preparation of Magnetic Beads Coated

With Anti-H2-DM Antibody

1. Place 2.0 x 108 Dynabeads labeled with sheep anti-rabbit IgG (DYNAL, cat. no. 112.03) into a 1.5-mL Eppendorf tube.

2. Place the tube on a magnet for 2 min and remove the fluid portion.

3. Remove the tube from the magnet and resuspend the beads in 0.8 mL of 1% BSA, 0.02% NaN3 in PBS.

4. Repeat steps 2 and 3.

5. Resuspend the beads in 60 |g of anti-H2-DM antibody in PBS containing 0.02% NaN3 (according to the manufacturer's protocol, approx 0.8-3.0 |g of antibody per 107 beads will give the necessary concentration to effectively bind a specific target). Make the final volume up to 0.8 mL with 1% BSA, 0.02% NaN3 in PBS.

6. Rotate the tube overnight in a refrigerator.

7. Place the tube on a magnet for 2 min and remove the fluid.

8. Remove the tube from the magnet and suspend the pellet in 0.8 mL of 1% BSA, 0.02% NaN3 in PBS.

9. Repeat step 8 and resuspend the beads in 0.5 mL of 1% BSA, 0.02% NaN3 in PBS. The final concentration should be 4.0 x 108 beads/mL.

3.4.2. Subcellular Fractionation of Vesicles Using Magnetic Beads Coated With Anti-H2-DM Antibody

1. Culture TEC line cells in 100-mm dishes with 10% FCS-JMEM.

2. Upon reaching confluency, culture the TEC line cells with 8 mL of 5% FCS-DMEM containing 100 IU/mL of IFN-y for 3 d. Replace the culture medium with the same volume of fresh medium every day.

3. After stimulation with IFN-y, remove the medium and rinse the dishes with 4 mL of 0.02% EDTA in PBS.

4. Remove the 0.02% EDTA and rinse with 4 mL of 0.25 M sucrose solution.

5. Remove the 0.25 M sucrose solution and replace with 0.8 mL of 0.25 M sucrose solution containing a protease inhibitor (1 mM PMSF, 0.1 mM TPCK, 10 |g/mL of aprotinin, 0.1 mM leupeptin).

6. Remove the cells using a cell scraper.

7. Transfer the cell suspension into a 15-mL test tube.

8. Put 0.8 mL of 0.25 M sucrose solution containing protease inhibitors into the dishes, and scrape the cells again.

9. Transfer the cell suspension into a 15-mL test tube and homogenize the cells by passing 1.6 mL of the 0.25 M sucrose solution 20 times through a 23-gauge needle, attached to a disposable 1-mL plastic syringe.

10. Centrifuge the homogenate at 1000g for 10 min to eliminate large cell fragments and nuclei.

11. Divide the supernatant (postnuclear supernatant, PNS) into two aliquots. Put each aliquot into a 1.5-mL screw cap tube.

12. Add 50 |L of the magnetic beads conjugated with the anti-H2-DM antibody (2 x 107 beads bound to approx 6 |g of antibody) into each aliquot of the PNS.

13. Rotate the tubes gently in a refrigerator for 24 h.

14. Place the tubes on a magnet for 10 min at 2-8°C and carefully pipet off the fluid.

15. Resuspend the pellet in 0.4 mL of PBS and mix gently.

16. Repeat steps 14 and 15.

17. Place the tubes on a magnet for 10 min at 2-8°C and carefully remove the fluid.

18. Store the pellet at -80°C until use.

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