Vesicle count Electron micrograph #Subject #1Subject #2Subject #3Subject #4Subject #5Subject #6 11016147105 27991374 397101271 471013885 557111387 68910968 71216512510 861310 45 91151316125 1051441277 1186137106 1281041987 1387 777 1411671769 1510 8 59 161589783 1741091287 1851210 77 197711785 20591011128 Supplementary Table 1. Exosome counting. Exosomes from six different normal subjects were used for this procedure. Five fields located in the center, upper right and left, and lower right and left of the grid were selected for viewing the exosomes from each subject. Four electron micrographs in each selected field were randomly taken at 20,000X magnification. Under this magnification the exosomes were clearly identifiable by their size, between 20 and 100 nm, and their round shape . Twenty electron micrographs were taken for each subject, therefore a total of 120 were taken for the entire procedure. A sampling error of ± 9% was estimated for this procedure based on the following formula: sampling error at 95% confidence = 98/sqrt(n), n = 120 in this case.
DTT (mg/ml) 050100200050100200 Pre - DTT treatmentPost - DTT treatment A DTT (mg/ml) 050100200050100200 Pre - DTT treatmentPost - DTT treatment B DTT (mg/ml) 050100200050100200 Pre - DTT treatmentPost - DTT treatment C D DTT 0 50 100 200 (mg/ml) * Supplementary Figure 1. Coomassie blue-stained SDS-PAGE gels of 17,000 Xg pellets before and after the treatments with different concentrations of DTT for 5 minutes at 37ºC. In order to optimize concentration of DTT treatment, 17,000 Xg pellets were resuspended and incubated with 0, 50, 100, or 200 mg/ml DTT at 37°C for 5 minutes followed by recentrifugation at 17,000 Xg. Gel loading was based on the same percentage of the pellets final volume. A, B, and C show results from three different experiments. THP is seen as a broad band around 92 kDa. D shows the average percentage (± SE) of THP present in the 17,000 Xg pellets after the DTT treatments compared to the 17,000 Xg pellets before the DTT treatments (combined data from experiment A, B, and C). The incubation of 17,000 Xg pellets with 200 mg/ml DTT significantly reduced the amount of THP in the 17,000 Xg pellets (*p = 0.03). 92 kDa - percentage
A 953760 215 17K Temperature (ºC) Time (min) 200K 215 953760 Aquaporin-2 B 37455060 5555 17K Temperature (ºC) Time (min) 200K 5555 Aquaporin-2 37455060 Supplementary Figure 2. Aquaporin-2 (AQP2) immunoblots of 17,000 Xg and 200,000 Xg pellets after incubation of the 17,000 Xg pellets with 200 mg/ml DTT at different temperatures and incubation times. In order to optimize temperature and incubation time for DTT treatment, 17,000 Xg pellets were incubated with 200 mg/ml DTT at different temperatures (37, 45, 50, 60, or 95°C) and incubation times (2, 5, or 15 minutes). Gel loading was based on the same percentage of the pellets final volume. In the 17,000 Xg pellet samples (the first four lanes in A and B), the signal intensity of an exosomal protein AQP2 in each lane is almost negligible indicating that, in all conditions, the DTT treatment successfully released the entrapped exosomes from THP polymeric network. In the 200,000 Xg pellet samples containing exosomes (the last four lanes in A and B), the samples that its 17,000 Xg pellet was incubated with DTT at 37ºC for 15 minutes (lane 6 in A) or at 37, 45, or 50ºC for 5 minutes (lane 5, 6, and 7 in B) show minimal aggregation and lack of degradation of AQP2. Thus 37ºC incubation was used in the subsequent experiments (Supplementary Figure 3). Lane 1 2 3 4 5 6 7 8
51015 17K 51015 200K Alix 37ºC 51015 17K 51015 200K Aquaporin-2 Supplementary Figure 3. Alix and aquaporin-2 immunoblots of 17,000 Xg and 200,000 Xg pellets after incubation of the 17,000 Xg pellets with 200mg/ml DTT at 37ºC for 5, 10, or 15 minutes. In order to optimize incubation time for DTT treatment, 17,000 Xg pellets were incubated with 200 mg/ml DTT at 37ºC for 5, 10, or 15 minutes. Gel loading was based on the same percentage of the pellets final volume. The band intensity values of the two exosomal proteins Alix (A) and aquaporin-2 (B) in the 200,000 Xg pellets (exosomal fraction) are very similar between the different incubation times. Time (min) 37ºC Time (min) AB
Protocol: Urinary exosome isolation by differential centrifugation (LKEM, NIH June 2009) Materials - Beckman® polycarbonate centrifuge tubes: #355655 with cap (capacity 70 ml) or #355630 (capacity 13.5 ml). Make sure that the tubes are clean. - A Beckman L8-70M ultracentrifuge with 45Ti or 70Ti rotor. Solutions Urine collection solution (volume per 50 ml of urine) o 1.67 ml 100 mM NaN3 (MW 65) o 2.5 ml AEBSF (2.75 mg/ml in ddH 2 O, stable 4°C for several weeks) o 50 µl Leupeptin (1 mg/ml in ddH 2 O, stable 1 wk 4°C, 6 mo -20°C) Isolation solution (prepared 50 ml) o 10 mM Triethanolamine (MW 185.7) 0.093 g o 250 mM Sucrose (MW 342.3) 4.28 g o ddH 2 O to 45 ml o Adjust pH to 7.6 with 1N NaOH ~ 220 µl o Add ddH 2 O to 50 ml Sample collection - Collect urine sample in a sterile container and add an appropriate amount of the urine collection solution. Differential centrifugation 1. 17,000 Xg centrifugation: step to remove whole cells, large membrane fragments, and other debris. - Add 55-65 ml of urine to #355655 tube (or 8ml of urine to #355630 tube) and centrifuge in a Beckman L8-70M ultracentrifuge with 45Ti rotor (or 70Ti rotor) at 17,000 Xg for 10 min at 37°C. - Keep the supernatant in a clean container at room temperature. This is supernatant #1. - Resuspend the pellet with the isolation solution, use approximately 500-800 µl for the pellet from #355655 tube or 50-100 µl for the pellet from #355630 tube (a volume to add depends on size of pellet). Add DTT to the resuspended pellet sample to a final concentration of 200 mg/ml. - Incubate the sample in a water bath or heat block at 37°C for 5-10 min, during incubation vortex the sample every 1-2 min, until the pellet is dissolved. - Transfer the sample to #355630 tube, add the isolation solution up to 8 ml, mix and then centrifuge the sample at 17,000 Xg for 10 min at 37°C (use 70.1Ti rotor). - Keep the supernatant in a clean container. This is supernatant #2. 2. 200,000 Xg centrifugation: step to pellet exosomes. - Pool the supernatant #1 and #2 in a clean centrifuge tube (depending on the volume, use either #355630 or # 355655 tube) and centrifuge in a Beckman L8-70M ultracentrifuge at 200,000 Xg. o For #355655 tube: use 45 Ti rotor, centrifuge at 42,200 rpm for 1 h and 54 min at 37°C. o For #355630 tube: use 70.1Ti rotor, centrifuge at 47,500 rpm for 1 h at 37°C. 3. Resuspension of exosome pellet - Discard the supernatant. - For immunoblotting, resuspend the pellet in 1.5% SDS and 50 mM Tris.HCl pH 6.8 (a volume to add depends on size of pellet). For electron microscopy, resuspend the pellet in 1X PBS.