ExoQuick-TC ™ is a proprietary polymer that gently precipitates exosomes and microvesicles between 30 and 200 nm in size of tissue culture media, urine or cerebrospinal fluid. First, pre-clear your cell samples and cell debris, and then simply add the appropriate amount of ExoQuick-TC to your cleaned biofluid, refrigerate and centrifuge. Your exosomes will be in the pellet, ready for resuspension in an appropriate solution.
ExoQuick-TC ™ Kits are shipped at room temperature, blue ice or dry ice and should be stored at + 4 ° C or at room temperature upon receipt. Properly stored kits are stable for 1 year from the date of receipt.
- The size of the reaction is based on the use of 5 ml of tissue culture medium or urine for the isolation of exosomes. For a better recovery for both RNA and protein analysis, we recommend starting with a 10 ml sample.
- To isolate exosomes from serum, we recommend using the ExoQuick reagent (cat # EXOQ5A-1 or EXOQ20A-1) which is a different formulation of the ExoQuick-TC reagent detailed in this manual.
- To isolate exosomes from plasma, we recommend using ExoQuick Plasma Prep and Exosome Precipitation Kit (Cat # EXOQ5TMA-1). Plasma contains fibrin which will precipitate together with ExoQuick causing an insoluble pellet to form. The ExoQuick Plasma Prep and Exosome Precipitation Kit contain reagents to help dissolve the fibrin precipitated, thus increasing the yield of exosomes.
Protocol: ExoQuick-TC ™
Note: this protocol is a starting point for most cells in culture. Different types of cells secrete different amounts of
exosomes. You may need to scale this protocol accordingly.
- Collect biofluid and centrifuge at 3000 × g for 15 minutes to remove cells and cell debris.
- Transfer the supernatant to a sterile container and add the appropriate volume of ExoQuick-TC to the biofluid. Mixture either by inverting or moving the tube.
- Refrigerate overnight (at least 12 hours) at + 4 ° C. Tubes should not be rotated or mixed during batching incubation period and should remain upright.
- Centrifuge the ExoQuick-TC / biofluid mixture at 1500 × g for 30 minutes. Centrifugation can be performed at
room temperature or + 4 ° C with similar results. After centrifugation, exosomes may appear beige or white balls in the bottom of the container.
- Aspirate the supernatant. Centrifuge the residual ExoQuick-TC solution by centrifugation at 1500 xg for 5 minutes. Remove all traces of liquid by aspiration, taking great care not to disturb the precipitated exosomes in the sediment.
- Resuspend the exosome pellet in 100-500 µl using 1X sterile PBS or specific buffer according to your
subsequent application. We recommend using the precipitated exosomes immediately rather than freezing them for future use.
Sample data and applications
The NanoSight LM10 instrument is based on a conventional light microscope and uses a laser light source to illuminate nanoscale particles within a 0.3 ml sample introduced into the display unit with a disposable syringe. Improved by a close perfect black background, the particles appear individually as point scatterers moving under Brownian motion. For NanoSight analysis, 2 ml of ExoQuick-TC was combined with 10 ml of Human HT1080 conditioned medium lung sarcoma cells or human embryonic kidney cells (HEK293).
5 ml of normal human urine was combined with 2.5 ml of ExoQuick-TC. All samples were incubated overnight at 4 ° C for exosome precipitation. The exosomes were resuspended in 1 ml of PBS and visualized on the NanoSight LM10 instrument (HT1080 culture media was diluted 1:40 and urine sample diluted 1:50 before analysis). Analysis of the HT1080 culture medium showed that ExoQuick-TC isolated 133 nm (peak) exosomes with a recovery of 1.74 X 109 particles/ml. HEK293 showed exosomes of 137nm with a recovery of 3.33X108 particles/ml. Normal human urine showed 107 nm exosomes with a recovery of 4.8 x 109 particles/ml.
2. Analysis of urine exosome protein markers
Ten millilitres of normal human urine were combined with 2 ml of ExoQuick-TC to precipitate urinary exosomes. The exosome pellet was resuspended with 175 µl of buffer and increasing amounts of the exosome suspension were loaded onto a plate prepared for ELISA. The CD9 protein was detected using the SBI rabbit anti-CD9 primary antibody and conjugated to SBI HRP goat anti-rabbit secondary antibody. The size of CD9 proteins in urine was determined by Western blot analysis with the same set of antibodies.