ExoQuick-TC User Manual
Contents
1. Ko ExoQui ck TC Exosome SBI isolation from Media and Urin System Biosciences ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 User Manual Store kit at 25 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 6 2015 09 01 contained in this user manual ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 Contents l Over ve R jh j OC O Oa ee 2 Il ProtoCOl oo 3 A Exosome precipitation ss 3 B RNA extraction from exosome o 4 C Protein extraction from exosome o 5 ELISA analysis 5 Western blotting sss 5 Il Sample Data and Applications sisi 7 IV References see 10 V Technical Support m j C CO 12 Vi Licensing and Warranty Statement 13 List of Components Reactions Catalog EXOTC50A 1 ExoQuick TC exosome precipitation solution 50 ml reactions The ExoQuick TC kits are shipped at room temperature or on blue ice and should be stored at 4 C upon receipt Properly stored kits are stable for 1 year from the date received The reaction size is based on using 5 ml of tissue culture media or urine for exosome isolation Examples of precipitating exosomes from various Biofluids can be seen in the Table below For best recovery for both RNA and Protein analysis we recommend starting with 10 ml sample Biofluid Samp2. of buffer Please see the next section of this protocol to determine the appropriate buffer for protein or RNA analysis 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual B Using Precipitated Exosomes for RNA Extraction For RNA extraction we recommend following the protocol outlined in the SeraMir Kit user manual as shown here Catalog RA800A 1 RA805A 1 RA806A 1 RA810A 1 and RA820A 1 1 If frozen thaw culture media or urine sample on ice 2 Combine 10ml sample 2ml ExoQuick TC Exosome 3 Mix well by inversion three times Isolation 4 Place at 4 C for 6 hours to overnight and Lysis 5 Centrifuge at 1500 x g for 30 minutes 6 Remove supernatant keep exosome pellet 7 Add 350 pl LYSIS Buffer to exosome pellet and vortex 15 seconds 8 Place at room temperature for 5 minutes to allow complete lysis optional add 5 ul of SeraMir control RNA spike in cat RA805A 1 9 Add 200ul of 100 Ethanol vortex 10 seconds 10 Assemble spin column and collection tube 11 Transfer all 600u to spin column 12 Centrifuge at 13 000 rpm for 1 minute exoRNA check to see that all flowed through Purification otherwise spin longer 13 Discard flow through and place spin column back into collection tube 14 Add 400l WASH Buffer 15 Centrifuge at 13 000 rpm for 1 minute 16 Repeat steps 13 to 15 once again total of 2 Washes 17 Discard flow through and centrifuge at
3. information on using the NanoSight instrument for exosome analysis visit http www nanosight com B Urine Exosome Marker Protein Analysis ExoQuick TC Urine Exosomes CD9 ELISA Assay R 0 9812 OD450 2 g g CD9 22 kDa 0 5 10 15 20 25 30 35 40 45 Amount of Urine exosomes added ul Ten milliliters of normal human urine was combined with 2ml ExoQuick TC to precipitate urine exosomes The exosome pellet was resuspended 175 ul buffer and increasing amounts of the exosome suspension was loaded onto an ELISA ready plate The CD9 protein was detected using Page 8 ver 6 2015 09 01 www systembio com ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 SBs rabbit anti CD9 primary antibody and SBI s HRP conjugated secondary goat anti rabbit antibody The size of urine CD9 proteins was determined using Western blot analysis with the same set of antibodies see inset C Activity Assays Track Exosomes using Cyto Tracers SBI has created a line of lentivector based Cyto Tracers that utilize GFP fusion proteins to mark cellular compartments organelles vesicles and structures to enable more long term and more in depth experimentation The Cyto Tracers can be used in transfections as well as packaged into virus to create stable GFP tracer cell lines in primary cells tumor cell lines and stem cells The Tetraspanin CD63 protein is a common biomarker for exosomes With the pCT CD63 GFP con
4. sample introduced to the viewing unit with a disposable syringe Enhanced by a near perfect black background particles appear individually as point scatterers moving under Brownian motion The image analysis Nanoparticle Tracking Analysis NTA software suite allows users to automatically track and size nanoparticles on an individual basis Results are displayed as a frequency size distribution graph Media from HT1080 cells Media from HEK293 cells Human Urine wetewe se ews om resse i W Ree ow Commies aot Oe Prete ts eee n Portate tse eee 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual For the NanoSight analysis 2ml of ExoQuick TC were combined with 10ml of conditioned media from Human HT1080 lung sarcoma cells or Human embryonic kidney HEK293 cells 5ml 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 1ml of PBS and visualized on the NanoSight LM10 instrument The HT1080 culture media were diluted 1 40 and the urine sample diluted to 1 50 prior to analysis HT1080 culture media analysis showed that ExoQuick TC isolated 133nm peak exosomes with a recovery of 1 74 X10 particles ml The HEK293 showed 137nm exosomes with a recovery of 3 33X10 particles ml Normal human urine showed 107nm exosomes with a recovery of 4 8X10 particles ml For more
5. temperature for 20 minutes to liberate exosome proteins 10 Centrifuge at 1500 x g for 5 minutes to remove all residual precipitation solution 11 Transfer supernatant to new centrifuge tube on ice 12 Exosome protein is now ready for immobilization onto micro titer plate Exosome Isolation and immobilization NEO OT Go Ne Please refer to the ExoELISA manual for the complete protocol Western blot analysis For Western blotting analysis we recommend resuspending the exosome pellet in 1XRIPA buffer with the appropriate protease inhibitor cocktail SBI offers a Western blot ExoAb Antibody Sampler Kit Cat EXOAB KIT 1 which includes four exosomal marker antibodies CD63 CD9 CD81 HSP70 and a Goat anti Rabbit IgG HRP conjugated secondary antibody specifically tested for use in exosomal protein analysis If frozen thaw culture media or urine sample on ice Combine 10 ml sample 2 ml ExoQuick TC Exosome Mix well by inversion three times Isolation and Place at 4 C for overnight at least 12 hours lysis Centrifuge at 1500 x g for 30 minutes Remove supernatant keep exosome pellet Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet NOaPaAN gt 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 11 12 13 14 15 16 17 18 Page 6 Add 200 ul RIPA buffer to exosome pellet and vortex 1
6. 13 000 rpm for 2 minutes to dry IMPORTANT Discard collection tube and assemble spin column with a fresh exoRNA RNase free 1 5ml elution tube not provided Elution Add 30u ELUTION Buffer directly to membrane in spin column Centrifuge at 2 000 rpm for 2 minutes loads buffer in membrane Increase speed to 13 000 rpm and centrifuge for 1 minute elutes exoRNAs You should have recovered 30 40yl exosome RNA The yield of RNA from isolated exosomes is different depending on the starting biofluid or the type of cells that were grown in culture Different cell types secrete varying levels of exosomes Page 4 ver 6 2015 09 01 www systembio com ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 C Using Precipitated Exosomes for Protein Extraction ELISA analysis SBI offers three ELISA kits Catalog ExoELISA 63 ExoELISA 9 ExoELISA 81 for fast and quantitative analysis of well characterized exosomal protein markers CD63 CD9 and CD81 If frozen thaw culture media or urine sample on ice Combine 10ml sample 2ml ExoQuick TC Mix well by inversion three times Place at 4 C for overnight at least 12 hours Centrifuge at 1500 x g for 30 minutes Remove supernatant keep exosome pellet Centrifuge at 1500 x g for 5 minutes to remove all traces of fluid take great care not to disturb the pellet 8 Add 200 ul Exosome Binding buffer to exosome pellet and vortex 15 seconds 9 Incubate at 37 C
7. 5 seconds Place at room temperature for 5 minutes to allow complete lysis Add Laemmli buffer with Beta mercaptoethanol and heat at 95 C for 5 minutes Chilled on ice for 5 minutes before loading onto gel Perform standard SDS PAGE electrophoresis and Western transfer onto PVDF membrane Block with 5 dry milk in Tris Buffered Saline 0 05 Tween TBS T for 1 hour Incubate blot overnight at 4 C with SBI s exosome specific antibody e g CD9 at 1 1000 dilution 5 dry milk in TBS T Wash 3X with TBS T Incubate one hour at room temperature with SBI s Goat anti Rabbit HRP antibody at 1 20 000 dilution 5 dry milk in TBS T Wash 3X with TBS T Incubate blot with chemi luminescence substrate and visualize on film or other imaging equipment 1X RIPA buffer contains 25mM Tris HCl pH 7 6 150mM NaCl 1 NP 40 1 sodium deoxycholate 0 1 SDS 2X Laemmli buffer contains 4 SDS 20 glycerol 10 2 mercaptoethanol 0 004 bromphenol blue 0 125 M Tris HCl pH 6 8 ver 6 2015 09 01 www systembio com ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 Q ExoAbs gt gt of OO P99 DD OSI NE oo og kDa 9 S 39 100 MW F 70 55 35 25 ExoQuick Exosome Serum Western Analysis lll Sample data and applications A NanoSight The NanoSight LM10 instrument is based on a conventional optical microscope and uses a laser light source to illuminate nano scale particles within a 0 3 ml
8. all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms ExoQuick exosome isolation methods are a patented technology Antes T et al Methods for Microvesicle Isolation and Selective Removal Patent No US 9 005 888 B2 The process of manufacturing of Exo FBS is a patented method in Patent No US 9 005 888 B2 The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility f
9. en BS Stirewalt DL Gentleman R Vessella RL Nelson PS Martin DB Tewari M Circulating microRNAs as stable blood based markers for cancer detection Proc Natl Acad Sci U S A 2008 Jul 29 105 30 10513 8 Laterza OF Lim L Garrett Engele PW Vlasakova K Muniappa N Tanaka WK Johnson JM Sina JF Fare TL Sistare FD Glaab WE Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury Clin Chem 2009 Nov 55 11 1977 83 Valadi H Ekstr m K Bossios A Sj strand M Lee JJ L tvall JO Exosome mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells Nat Cell Biol 2007 Jun 9 6 654 9 Pegtel DM Cosmopoulos K Thorley Lawson DA van Eijndhoven MA Hopmans ES Lindenberg JL de Gruijl TD Wordinger T Middeldorp JM Functional delivery of viral miRNAs via exosomes Proc Natl Acad Sci USA 2010 Apr 6 107 14 6328 33 Mathivanan S and Simpson R J ExoCarta A compendium of exosomal proteins and RNA Proteomics 2009 21 4997 5000 Page 10 ver 6 2015 09 01 www systembio com ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 Thery C Ostrowski M Segura E Membrane vesicles as conveyors of immune responses Nat Rev Immunol 2009 8 581 93 Michael A Bajracharya SD Yuen PS Zhou H Star RA Illei GG Alevizos Exosomes from human saliva as a source of microRNA biomarkers Oral Dis 2010 Jan 16 1 34 8 Luo SS Ishibashi O Ishikawa G Is
10. hikawa T Katayama A Mishima T Takizawa T Shigihara T Goto T Izumi A Ohkuchi A Matsubara S Takeshita T Takizawa T Human villous trophoblasts express and secrete placenta specific microRNAs into maternal circulation via exosomes Biol Reprod 2009 Oct 81 4 717 29 Taylor DD Gercel Taylor C MicroRNA signatures of tumor derived exosomes as diagnostic biomarkers of ovarian cancer Gynecol Oncol 2008 Jul 110 1 13 21 Simpson Ru Lim JW Moritz RL Mathivanan S Exosomes proteomic insights and diagnostic potential Expert Rev Proteomics 2009 Jun 6 3 267 83 Review Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual VI Licensing and Warranty Statement Limited Use License Use of the ExoQuick TC Exosome Precipitation Solution i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return
11. icrovesicle Isolation and Selective Removal Patent No US 9 005 888 B2 The process of manufacturing of Exo FBS is a patented method in Patent No US 9 005 888 B2 ver 6 2015 09 01 www systembio com ExoQuick TC Exosome Precipitation Solution Cat EXOTCxxA 1 PROTOCOL A Exosome Precipitation 10 ml starting sample Isolate exosomes with ExoQuick TC 1 Collect biofluid and centrifuge at 3000 x g for 15 minutes to remove cells and cell debris 2 Transfer supernatant to a sterile vessel and add the appropriate volume of ExoQuick TC Exosome Precipitation Solution to the Biofluid Some examples are shown in the Table below Mix well by inverting or flicking the tube Incubation Biofluid Sample volume ExoQuick TC Time 12 hours Overnight volume Urine 12 hours Culture Overnight media 3 Refrigerate overnight at least 12 hours The tubes do not need to be rotated during the incubation period 4 Centrifuge ExoQuick TC biofluid mixture at 1500 x g for 30 minutes Centrifugation may be performed at either room temperature or 4 C with similar results After centrifugation the exosomes may appear as a beige or white pellet at the bottom of the tube 5 Aspirate supernatant Spin down residual ExoQuick TC solution by centrifugation at 1500 x g for 5 minutes Remove all traces of fluid by aspiration taking great care not to disturb the precipitated exosomes in pellet 6 Resuspend exosome pellet in 100u 500u
12. le volume ExoQuick TC volume Urine 5 ml imi Spinal fluid 5 ml 1 ml Culture media 5 ml 1 ml For best RNA and Protein recovery 10ml sample Urine 10 ml 2 ml Spinal fluid 10 ml 2 mi Culture media 10 ml 2 mi 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual ExoQuick TC Exosome Precipitation l Overview Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro Exosomes are found in blood urine amniotic fluid malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted SBI s ExoQuick TC exosome precipitation reagent makes microRNA and protein biomarker discoveries simple reliable and quantitative Enrich for exosomal microRNAs with ExoQuick TC and accurately profile them using SBI s SeraMir qPCR arrays Downstream protein analysis is also possible with SBI s exosome specific antibodies and ELISA kits No time consuming ultracentrifugation Less expensive than costly antibodies and beads More effective than any other method Use as little as 5 ml media or urine samples Page 2 ExoQuick TC ya Exosome isolation from Media and Urin Media from HT1080 cells NanoSight Analysis LPA o 100 pee tate ee Cenaa hae j TRIO ExoQuick exosome isolation methods are a patented technology Antes T et al Methods for M
13. or injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2015 System Biosciences SBI All Rights Reserved Page 12 ver 6 2015 09 01 www systembio com
14. struct you can make you cells of interest secrete exosomes that glow green for downstream functional delivery studies Cat CYTO120 PA 1 Transduced 293 cells pCT CD63 GFP CD63 Tetraspanin Tag _ Catalog CYTO120 PA 1 gt A bd a A L tA wwe P f ms a Phase Transfected 293 cells 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual IV Citations As featured in Exosome Isolation for Proteomic Analyses and RNA Profiling Douglas D Taylor Wolfgang Zacharias and Cicek Gercel Taylor Serum Plasma Proteomics Methods in Molecular Biology 2011 Volume 728 Part 4 235 246 PDF Tae Hoon Lee Esterina D Asti Nathalie Magnus Khalid Al Nedawi Brian Meehan and Janusz Rak Review Microvesicles as mediators of intercellular communication in cancer the emerging science of cellular debris Seminars in Immunopathology DOI 10 1007 s00281 011 0250 3 PDE Technical References Adachi T Nakanishi M Otsuka Y Nishimura K Hirokawa G Goto Y Nonogi H Iwai N Plasma microRNA 499 as a biomarker of acute myocardial infarction Clin Chem 2010 Jul 56 7 1183 5 De Smaele E Ferretti E Gulino A MicroRNAs as biomarkers for CNS cancer and other disorders Brain Res 2010 Jun 18 1338 100 11 Mitchell PS Parkin RK Kroh EM Fritz BR Wyman SK Pogosova Agadjanyan EL Peterson A Noteboom J O Briant KC Allen A Lin DW Urban N Drescher CW Knuds
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