Exosome IsolationDiscover RNA and Protein Biomarkers in Patient Biofluids and Culture Media
• Simple one-step precipitation
• No time-consuming ultracentrifugation
• No complicated syringes required
• Less expensive than costly antibodies and beads
• More effective than any other method
• Isolate intact exosomes for functional studies as well as microRNA and proteins
• Compatible with biofluid from any species
• Use as little as 250 µl of serum, plasma (see also special kit), or tumor ascites fluid, or 5 ml of media, spinal fluid, or urine
• Exosome depleted FBS available to avoid bovine derived exosomes in your experiments
Exosomes are 40 –100 nm membrane vesicles secreted by most cell types in vivo and in vitro. Their cargo reflects the origin and pysiological stet of the source cells.
Exosomes are found in blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs depending upon the tumor from which they are secreted. SBI´s ExoQuick and ExoQuick-TC polymer-based exosome precipitation reagents make microRNA and protein biomarker discoveries simple, reliable and quantitative.
Circulating exosomes isolated by ExoQuick and ExoQuick-TC yield exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation or DynaBeads. This enhances the senitivity and accuracy of downstream applications, such as qRT-PCR Profiling of miRNA and mass spectrometric or electrophoretic analyses of exosomal proteins.
Enrich for circulating exosomal microRNAs with ExoQuick or ExoQuick-TC (depending on the sample type) and accurately profile them using QuantiMir qPCR arrays.
Exosome Precipitation using ExoQuick
Isolation of Exosome microRNAs from Ovarian Tumor Ascites Fluid
Exosomes were isolated with different methods as indicated.
Exosomal RNA was purified and RNA quality and yield was accessed using a GeneQuant II. Small RNAs were analyzed with the Agilent 2100 Bioanalyzer Lab-on-a-Chip instrument system (Agilent Technologies, Santa Clara, CA), using the Agilent Small RNA chip and reagent kit. Approximately 100ng of isolated total RNA in 1μl was applied to each run. The manufacturer’s recommended protocol was strictly followed to obtain Bioanalyzer profiles for the size range 6 to 150 nucleotides (nt). The profiles were calibrated for size (nt) using the small RNA ladder supplied with the kit, containing markers of 20, 40, 60, 80, and 150 nt in size, as reference. The instrument software quantitated the peak area between 0 and 150 nt as small RNA region, the area within 10 to 40 nt as microRNA region, and provides percentages of miRNA detected for each sample.
Isolation of Exosome Proteins from Ovarian Tumor Ascites Fluid
Enrichment of Placental Alkaline Phosphatase
Exosomes were isolated with different methods as indicated.
The quantity of protein was determined by the Bradford microassay method (Bio-Rad Laboratories), using BSA as a standard. Proteins from each exosome isolate were standardized to the original sample volume and equal volumes were applied per lane of a 12.5% SDS-PAGE gel. Western immunoblotting was performed to analyze the presence of the specific marker protein, placental alkaline phosphatase (PLAP). The SDS-PAGE gel was transferred to a nitrocellulose membrane, the membrane blocked for 1 hour at room temperature with non-fat dried milk, and probed overnight at 4°C with primary antibody. The bound immune complexes were visualized by enhanced chemiluminescence (ECL, Amersham Life Sciences) and quantitated by densitometry (Un-Scan-it Software, Silk Scientific Corp.).
Human Urine Exosome Protein Biomarker Analysis
Human urine samples (1 ml) were treated with 1 ml ExoQuick. Exosome pellets recovered were resuspended in 35µl PBS and the supernatants used as controls. Equal volumes (10µl) of urine supernatant (Sup) and exosome pellets (EXO) were separated on 4–15% gradient PAGE gels (Bio-Rad). Standard Western blot procedures with antibodies to Annexin V and Aquaporin-2 (both from Abcam, Inc.) were used to detect urine exosomal protein biomarkers.
ExoQuick isolated exosomes are functional
ExoQuick exosomes can be transfered between cells.
SBI created a stable 293TN cell line overexpressing the Cyto-Tracer™ pCT-CD63-GFP fusion protein (catalog# CYTO120-PA-1-SBI). The media from the cells was collected 48 h after plating and the exosomes from the media were precipitated using ExoQuick-TC. The exosome pellet recovered was resuspended in 30ul PBS and 10ul was added to newly plated HT1080 cells. HT1080 cells were visualized 72 h after the addition of the CD63-GFP labeled exosomes and then re-plated. Following another 24h, the cells were again visualized for GFP fluorescence and imaged. The exosomes appear to dock with the cells within 72 h and some are found to be internalized after 96 h.
New in the ExoQuick Product Line: ExoQuick-LP
Pre-clear Serum Lipoprotein Particles Before Exosome Isolation with the new kit ExoQuick-LP.
In order to study only the exosome-associated microRNAs and proteins, it is important to remove as many subcellular particles as possible from blood plasma or serum before the treatment with ExoQuick, particularly lipoprotein particles. Lipoproteins are the major lipid-based particles in the plasma and serum and it has been recently demonstrated that both high-density lipoproteins (HDLs) and low-density-density lipoproteins (LDLs) contain microRNAs (Vickers et al. 2011).
Scalable exosome isolation is possible now
The gold standard for isolating exosomes is an ultracentrifugation step, a technique that can be a barrier to high-throughput processing. Clearly, more scalable protocols that work on small sample volumes are needed as exosome analysis moves from research and development and into the clinic. SBI´s ExoQuick is such an alternative. With ExoQuick, exosomes can be isolated from as little as 100 μl of serum in just 2.5 hours, as can be seen in a recent paper from Khan, et al. (1)
Exo-FBS™ Exosome-depleted FBS
• Exosome-sized and CD63 vesicles removed
• No detectable cow exosomes by Western blot
• No detectable cow microRNAs by qPCR
• Same cellular growth rates supported as standard FBS
• Use the same as standard FBS (add 10% in DMEM or RPMI)
• Optimized for exosome research with numerous peer-reviewed publications
Standard growth medium for most cells in culture require fetal bovine serum (FBS) as a growth supplement to DMEM. FBS is derived from bovine (cow) serum and contains a high abundance of cow exosome vesicles. These exosomes can interfere or cause significant background issues when studying the exosomes secreted from your cells of interest in standard culture conditions.
Our partner SBI has developed an exosome-depleted FBS growth supplement called Exo-FBS that has been stripped of bovine exosomes. Exo-FBS supports equivalent growth of many types of cells in culture, contains very low levels of cow CD63 positive exosomes and does not have any measurable bovine microRNAs. Perform your studies of cellular secreted exosomes in culture without the worry of contaminating cow exosomes in your experiments. No ultracentrifugation required. Available as standard FBS supplement or heat inactivated FBS media supplement (treated at 65°C for 15 minutes before bovine exosome removal).
For more information see individual Exo-FBS products and product brochure (link below).
ExoQuick PLUS and ExoQuick-TC PLUS - Quick and Efficient Exosome Isolation for Sensitive Applications
...when you need an extra level of confidence:
• Cleaner—reduces albumins and immunoglobulins as much as 85% more than leading column-based serum exosome isolation kits
• Better—reduces total protein carryover as much as 50% more than competing column or precipitation-based approaches
• Powerful—detects biomarkers with as much as a 3-fold increase in sensitivity
• Fast—takes less than 10 minutes of hands-on-time after initial ExoQuick isolation
While the ExoQuick® family of reagents speed-up and simplify exosome isolation and are compatible with most downstream uses, some applications are especially sensitive to carry-over of non-exosomal proteins applications such as mass spectrometry, western blotting of low abundance markers, exosome labeling, and in vivo/ex vivo exosome delivery. Which is why SBI offers ExoQuick PLUS and ExoQuick-TC PLUS.
Building on SBI´s popular ExoQuick family of products, ExoQuick PLUS and ExoQuick-TC PLUS further reduce protein carryover from serum/plasma and tissue culture exosomal preparations with a simple treatment by ready-to-use, customized microspheres (kits contains enough ExoQuick or ExoQuick-TC and pre-packed microspheres for 10 reactions. In less than 10 minutes of total hands-on time after ExoQuick isolation, you can obtain highly pure, intact exosomes suitable for a variety of protein-sensitive applications.
For ExoQuick PLUS and ExoQuick-TC PLUS validation data and user manuals see individual products.
More Product Citations
NanoSight Data showing the exosome isolation with ExoQuick
Data showing biological activity of the purified exosomes
Taylor et al.: Abstract about Exosome Isolation
Combination Kit: ExoQuick plus exoRNA Purification Columns
Combination Kit: ExoQuick-TC & exoRNA Purification Columns
Exosomal microRNA Profiling from Serum
QuantiMir qPCR Arrays
Exosome Labeling using Cyto-Tracer GFP-Fusion Vector
Exocarta online database of exosome proteins and RNAs
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|ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (5 ml)||EXOQ5A-1-SBI||75 rxns (250 ul sample volume)||410 €||DETAILS||Add to Cart|
|ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (20 ml)||EXOQ20A-1-SBI||300 rxns (250 ul sample volume)||1083 €||DETAILS||Add to Cart|
|ExoQuick Exosome Precipitation Solution for Serum, Plasma and Tumor Ascites Fluid (100 ml)||EXOQ100A-1-SBI||1500 rxns (250 ul sample volume)||3974 €||DETAILS||Add to Cart|
|ExoQuick PLUS Exosome Purification Kit -Serum and Plasma (10 reactions)||EQPL10A-1-SBI||10 rxns||554 €||DETAILS||Add to Cart|
|ExoQuick Plasma prep and Exosome precipitation kit (5 ml ExoQuick plus 500 ul Thrombin)||EXOQ5TM-1-SBI||75 rxns (500 ul sample volume)||796 €||DETAILS||Add to Cart|
|Thrombin Plasma Prep for Exosome Precipitation (100 x Thrombin)||TMEXO-1-SBI||100 reactions||569 €||DETAILS||Add to Cart|
|ExoQuick-LP Exosome Lipoprotein Pre-Clear and Exosome Precipitation Kit||EXOLP5A-1-SBI||5 rxns||496 €||DETAILS||Add to Cart|
|ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (10 ml)||EXOTC10A-1-SBI||10 rxns (5 ml sample volume)||379 €||DETAILS||Add to Cart|
|ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (50 ml)||EXOTC50A-1-SBI||50 rxns (5 ml sample volume)||1089 €||DETAILS||Add to Cart|
|ExoQuick-TC Exosome Precipitation Solution for Culture Media, Spinal Fluid and Urine (250 ml)||EXOTC250A-1-SBI||250 rxns (5 ml sample volume)||3350 €||DETAILS||Add to Cart|
|ExoQuick-TC PLUS Exosome Purification Kit - Tissue Culture Media (10 reactions)||EQPL10TC-1-SBI||10 rxns||554 €||DETAILS||Add to Cart|
|ExoQuick-CG cGMP-grade Exosome Isolation reagent||EXOCG50A-1-SBI||30 rxns (5 ml sample volume)||1054 €||DETAILS||Add to Cart|
|Exosome-depleted FBS Media Supplement||EXO-FBS-50A-1-SBI||50 ml||298 €||DETAILS||Add to Cart|
|Exosome-depleted FBS Media Supplement||EXO-FBS-250A-1-SBI||250 ml||859 €||DETAILS||Add to Cart|
|Exosome-depleted FBS Media Supplement Heat Inactivated||EXO-FBSHI-50A-1-SBI||50 ml||308 €||DETAILS||Add to Cart|
|Exosome-depleted FBS Media Supplement Heat Inactivated||EXO-FBSHI-250A-1-SBI||250 ml||871 €||DETAILS||Add to Cart|
|FBS Exosome Depletion Kit I-Column Format||61200-NB||Deplete up to 120 ml FBS||307 €||DETAILS||Add to Cart|
|FBS Exosome Depletion Kit I-Slurry Format||61100-NB||Deplete up to 140 ml FBS||291 €||DETAILS||Add to Cart|
|FBS Exosome Depletion Kit II-Column Format||61300-NB||Deplete up to 240 ml FBS||530 €||DETAILS||Add to Cart|
|FBS Exosome Depletion Kit II-Slurry Format||61400-NB||Deplete up to 280 ml FBS||464 €||DETAILS||Add to Cart|