3.3.1. Western Blot Analysis
For Western blot, exosomes (3-10 pg) are separated by 12 or 15% sodium dodecyl sulfate-polyacrylamide gels, semidry transferred onto PVDF and detected by Western blotting using an enhanced chemiluminescence detection kit. Exosomal proteins for which we routinely blot include Hsc70, CD71, and major histocompatibility complex class II. The basic protocol is as follows:
1. Dilute the exosomes in 5X sodium dodecyl sulfate sample buffer.
2. Boil the samples for 5 min at 95°C and load onto a sodium dodecyl sulfate-polyacrylamide gels.
3. After the gel has finished running, set up the semidry transfer following the manufacturer's protocol.
4. After transferring, check for the transfer of proteins by staining the membrane with Amido Black. Destain the membrane by washing in MeOH for 3 x 5 min. Wash the membrane in PBS-T for 5 min.
5. Block the membrane for 1 h or overnight in 5% dry milk in PBS-T.
6. Wash the membrane in PBS-T for 3 x 5 min.
7. Incubate the membrane in primary MAb (in 1% BSA/PBS-T) for 2 h or overnight using the supplier's dilution recommendations.
8. Wash the membrane in PBS-T for 3 x 1 min, then 3 x 5 min.
9. Incubate the membrane in secondary antibody labeled with horseradish peroxidase (in 5% milk/PBS-T) at a dilution of 1:5000 for 1 h.
10. Wash the membrane in PBS-T for 3 x 1 min, once for 15 min, then 3 x 5 min.
11. Detect proteins using chemiluminescence kit of choice and develop on autoradi-ography film.
For flow cytometry, exosomes are required to be attached to latex beads that have been precoated with an antibody that recognizes markers exposed on the exosome surface. Then the exosome-latex bead complexes are labeled with fluorochrome-conjugated MAb. This method is necessary because of the small size of the exosomes (see Note 6).
1. Use 2.5 mL of 4-|im latex beads (4% solids) and wash with 10 mL of MES buffer.
2. Centrifuge the beads at 3000g for 20 min.
3. Repeat the washing and centrifugation steps.
4. Resuspend the beads in 5 mL of MES buffer.
5. Add 250 |L of beads (5 mg) to 250 |L of BSA in MES buffer (1 mg/mL) and 60 |g of antibody that will be used to attach the exosomes.
6. Incubate overnight at RT, rocking slowly.
7. Centrifuge at 3000g for 20 min.
8. Remove the supernatant and resuspend in 1 mL of PBS.
9. Repeat steps 7 and 8.
10. Centrifuge at 3000g for 20 min.
11. Remove the supernatant and resuspend in 500 |L of storage buffer.
12. To set up the experiment, distribute 15 |L of labeled beads into the desired number of microfuge tubes.
13. Add 50-100 |g of exosomes and make up the volume to 500 |L. Alternatively, a master mix can be made of beads and exosomes, then distributed into FACS tubes at step 16.
14. Incubate for 2-4 h at 4°C, rocking slowly.
15. Centrifuge at 700g in a microcentrifuge for 5 min.
16. Remove the supernatant and resuspend in 100 |L of cold HBSS or staining buffer, then distribute the beads to FACS tubes.
17. Block each tube with 10 |L of normal goat serum for 10 min on ice.
18. Add 1-2 |L of labeled antibody (e.g., FITC or PE). Incubate for 30-45 min on ice. Cover the tubes, as the fluorophores are light sensitive.
19. To wash away excess MAb, add 1 mL of cold HBSS or staining buffer to each tube. Centrifuge the tubes for 5 min at 700g.
20. Aspirate the supernatant and resuspend in 200-400 | L of staining buffer. If not analyzing immediately, resuspend in 200 |L of cold 4% paraformaldehyde.
21. Analyse by FACS.
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