Exosome isolation, Subcellular fractionation, and TCA precipitation

Exosome isolation using filtration and ultracentrifugation

1. Plate cells on 10 x 10cm dish and let grow until about 80% confluent.
2. Plate cells on 10 x 225cm³ flask on normal growing media and let grow 1 to 2 days until -about 80% confluent.
3. Aspirate growing media, wash cells 2 x with plain media or DPBS 
4. Add 45 to 50ml plain (no serum) media.
5. Let grow 72h.
6. Collect medias into 500ml centrifuge bottles.
   • Add plain media back into one flasks and put back in the incubator → harvest and lyse cells during 90 min ultracentrifugation steps using subcellular fractionation protocol (P1, P2, S2)
7. Cool down conditioned media from serum-free cell cultures on ice 
8. Centrifuge 350 g, 10 min
9. Pass through 0.2 μm pore filters. 
   • Add an inhibitor cocktail to protect the proteins from proteolytic digestion (Options: PMSF, Inhibitor cocktail complete™ Roche, Mannheim, Germany).
10. Ultrafiltrate using Amicon Ultra 100K, cut-off 100 kDa Millipore 25 min 4000g (10 x 15ml)
    • Repeat this step until all media has been filtrated. (you can use the same filters multiple time during the isolation)
11. Ultracentrifugate at 120,000 ×g for 90 min at 4 °C
12. Wash pellet with ice-cold PBS
13. Ultracentrifugate at 120,000 ×g for 90 min at 4 °C
14. Aspirate washing media ja resuspend on PBS (50ul to 200 ul, depending on the pellet size)
15. Make small aliquots and store in -80 freezer.
16. Measure protein concentration and validate by western blot and electron microscopy.

Subcellular Fractionation

1. Harvest cells and spin down 3000rpm 5min 4°C.
2. After harvesting the cells, all steps are done on ice to prevent proteolysis. 
3. Wash twice with cold 1x PBS, spin down.
4. Gently resuspend pellet in HNMEK lysis buffer and incubate on ice for 20 minutes. 
5. Following incubation, lyse the cells using glass douncer (20 strokes). 
6. Centrifuge the lysate 500g 10 minutes 4°C → collect pellet 1 (P1), nucleai
   • wash pellet with 500ul buffer and spin again
   • Resuspend in RIPA buffer.
     • Measure concentration and normalize 
   • Add 5x sample buffer and boil 5-10 min.
7. Centrifuge supernatant (S1) at 10000g for 10 minutes 4°C → collect pellet 2 (P2), organelles 
   • take small aliquot from supernatant, S2 fraction
   • wash pellet with 500ul buffer and spin again
   • Resuspend in RIPA buffer.
     • Measure concentration and normalize 
   • Add 5x sample buffer and boil 5-10 min.
8. Centrifuge supernatant 100 000g 90mins 4°C → collect pellet 3 (P3), membrane.
   • take small aliquot from supernatant, S3 fraction
   • wash pellet with 500ul buffer and spin again
   • Resuspend in RIPA buffer.
     • Measure concentration and normalize if needed
   • Add 5x sample buffer and boil 5-10 min.

HNMEK lysis buffer 

20 mM HEPES (pH 7.4) 

150 mM NaCl

1.5 mM MgCl₂

2 mM EDTA

10 mM KCl

0.5 mM EGTA

Before use add:

1 mM DTT 

1x protease inhibitor cocktail tablet from Roche

RIPA Buffer

50mM Tris, pH7.4

150mM NaCl

5mM EDTA

1% NP-40

0.5% DOC (sensitive)

0.1% SDS

Before use add:

1 mM PMSF

1x protease inhibitor cocktail tablet from Roche

TCA precipitation

Day 1

1. Collect ~20% of the flow through after ultrafiltration step during the exosome isolation
2. Add deoxycholate (sodium salt) to final concentration of 150 ug/ml, vortex and incubate on ice for 15 min. 
3. Add TCA (from -20°C) in the chemical hood to a final concentration of 8-10%. 
4. Vortex and place on ice – incubate at 4°C on ice O/N.
   • Proteins will precipitate

Day 2

1. Transfer 1.5 ml of O/N precipitation into a 1.5ml tube and spin down at max speed at 4°C for 15 min. 
2. Remove sup and repeat step 1 in the same tube until all the O/N volume is spinned down. 
3. Add 400ul of Acetone (from -20°C) and spin down at max speed, 4°C for 10 min (just a wash, do not resuspend pellet). 
4. Repeat step 3. 
5. After removing acetone from tubes, let air dry for 20 min on bench/hood until you don't see any traces of acetone
6. Re-suspend pellet in 20ul 10mM Tris (pH 8) (You will see pellet smear in the tube wall too)
7. Add 20ul 2x sample buffer (if turns yellow, add 0.5-1ul 1M NaOH) 
8. Boil sample 10 min.  
9. Load onto SDS-PAGE for western blot.

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