I have previously been attempting to pellet extracellular vesicles (EVs) from 1mL PBS resupension in Beckman 357448 1.5mL polyallomer tubes. However I keep getting DNA yields and Phi29 polymerase RCA product signals (radiometric DNA blot) which overall less that if I don't wash and are inconsistent (see diagram attached). I believe I am sucking off the pellet. My PBS wash spins are performed on basis of k-factor adjusted spin speeds when I switch from a SW 41 Ti rotor for my extraction spin to a TLA 110 for the 1.5mL PBS spins (see attached excel sheet). I perform my PBS wash spins for 5x longer than required to ensure I don't lose yield from incomplete pelleting.
Would using a marker dye be recommended to avoid sucking a pellet? I've tried using hematoxylin (1:1000 dilution) but it just precipitates regardless if there are EVs or not (see attached). I haven't noticed it inhibit either lysis or my RCA reaction and it precipitates in lysis buffer allowing me to readily remove it from my pellet lysate. Are there other options?
Passivation of the tubes with BSA or similar may help. Depending on what you plan to do with the exosomes after purification, staining the exosomes with a membrane dye (e.g. Cellmask Orange plasma membrane stain) lets you see the pellet and, more importantly, ensures that it is fully resuspended.
Hi,
-What's the speed of centrifugation you use ?
-What the time period of your centrifugation you use ?
-What's the temperature you use ?
-What cell's type are you trying to extract MV from ?
-On the picture you attached, it looks like a Falcon 12 mL tube. Do you use RNAse / DNAse treated material systematically ?
-What's your procedure for discarding the supernatant and resuspending MV pellet ?
The MV pellet is usually very difficult to suck off when the ultracentrifugation steps settings are properly applied.
Antoine
Try once 70 Ti or 50 Ti rotor and polycarbonate tubes. At least 1,20,000 g (around 34600 rpm) for at least 1:30 h. Use chilled filtered PBS and cool rotor prior to centrifuge. No brake and minimum decelerate (1-3 max). Takeout tubes immediately after centrifuge do not keep the rotor sit idol for long time (> 5-10 min). Mark the possible position using marker in the tube where the pellet expected to settle (as minute amount of pellet is not visible). Keep the tubes up side down in -20 C for sometime to locate the pellet (transparent whitish).
I have already use PKH26 to stain exosomes...It works very well and you can see the pink pellet perfectly. Just test if it could interfere with your experiments....
I use DDAO-SE to stain my vesicles, which binds to all membrane proteins, giving a dark blue pellet. This particular dye fluoresces in the far red channel at 605/640 so hopefully it won't affect your downstream applications.
I'm trying to extract Apoptotic Bodies, Microvesicles and Exosomes from immortalised cancer lines using differential ultracentrifugation. I use pre-chilled PBS for all my washing and keep tubes on ice during transport. For decceleration I use a setting of 8 (Beckman MX) or "slow" (Beckman L90K) on a SW 41 Ti for initial extraction spin. For wash spins, I use a TLA110 rotor with adaptor for 1.5mL centrifuge tubes (#357448). Please see Rotor Speed Calc excel sheet for rotor speeds/durations. I remove the PBS using pipette (facing away from where I think the pellet is) and get about 2 mm towards the bottom
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