Urine sample collection, storage, and processing procedures for isolation of urinary exosomes

• Sample collection (protease inhibitor) solution (volume per 50 ml urine)
• 1.67 ml of 100 mM sodium azide (NaN3)
• 2.5 ml PMSF (2 mg/ml in isopropyl alcohol, stable 4°C for several months)
• 50 µl Leupeptin (1 mg/ml in ddH2O, stable 1 week at 4°C, 6 months at -20°C)

• Collection
• If possible, determine the collection time and volume.
• Collect the first or second morning urine, 10~100 ml (See Notes 1 and 2).
• First morning urine: first urine after waking (no fluid or fruits after 9 PM the prior evening).
• Second morning urine: Discard the first urine. Collect the next voided urine. May have breakfast and undergo regular activity between first and second morning urine.
• Can also use random urine samples

• Storage
• Add sample collection (protease inhibitor) solution (see above)
• If possible, process samples immediately (refrigerate at 4 °C).
• If the urine samples cannot be processed immediately, the samples should be frozen at -80 °C (NOT -20 °C). (See Note 3)
• 50 ml of urine samples are aliquoted in 50 ml tubes and stored at -80°C as soon as possible after collection.

• Defrosting and processing
• Frozen urine samples should be thawed at room temperature (requires ?3 hrs for a 50 ml urine sample). Avoid prolonged thawing on ice (4 °C).
• While urine is defrosting (i.e., is still a mixture of ice and water), extensively and vigorously vortex for one minute.
• After sample completely thaws, vigorously vortex for an additional 30 sec, then proceed to differential centrifugation for urinary exosome isolation.
• Insufficient vortexing will result in major loss of urinary exosomes.

• Notes:
1. 10 ml of urine is sufficient to detect exosomal TSG101, NHE3, AQP2, and ALIX by western blotting.
2. The difference between first morning urine and second morning urine is very small. Either can be used for analysis of TSG101, NHE3, AQP2, and ALIX.
3. Freezing at -20 °C causes a major loss of urinary exosomes that can be partially restored by extensive vortexing.
4. In urine from healthy individuals, 1 ml of urine yields approximately 2 ul of 200,000 × g spin pellets. The concentration of urinary exosomal protein is ?1.1 ug/ul .
5. Western blot analysis: Mix equal volume of Laemmli sample buffer (Bio-Rad) including 60 mg/ml DTT to isolation solution, heat samples at 60 °C for 10 min, then store samples at -80 °C for western blot analysis.