Exo-Flow and IP Kits User Manual
Contents
1. Iron magnetite central layer Fig 1 Exo Flow streptavidin magnetic beads Depending upon the specific exosomes you wish to purify a particular biotinylated antibody may be used to couple to the Exo Flow streptavidin beads The Exo Flow kits are modular thus you can select from various pre validated capture antibody kits or utilize your own biotinylated capture antibody corresponding to the exosome surface marker specific for the exosomes of interest in your model system SBI has thoroughly tested a variety of capture antibodies that work quite well to flow sort exosomes from either serum or cell culture samples These are highlighted by arrows in the diagram below Page 2 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx There are a few categories of known exosome surface markers based upon some functional attributes Marker type Functions Examples Exosome formation CD9 CD63 ea epee and secretion 1 0D31 CD44 Immune modulation MHG Ill anergy and priming HLA G Exosome biogenesis SNAP secretion and Rab5b downstream cell fusion i i j j z of Pi W T2 Membrane transport and fusion Fig 2 Exosome surface marker examples The Exo Flow beads will appear as an orange or pumpkin like color which allows for them to be easily tracked during the magnetic separations and washes The beads adhere strongly after placed on the magnetic stand such that clean separat2. SBI System Biosciences Exo FLOW Exosome Purification Kits Cat s EXOFLOWxxx User Manual Store Kits at 4 C upon 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 contained in this user manual Exo Flow Exosome Capture Kits Cat EXOFLOWxxx Contents ke WPIOGUCIION eee eae 1 A EXOSOME OVEIVICW ahonnan a 1 B Magnetic Beads and Exosome Surface Markers 66 2 C The Exo FITC Universal and Reversible Stain 00 4 Dr Exo Flow FACS Protocols iicxreiat cn heielicte nek teehee 4 E Sample Exo Flow FACS data ccccccssssseeeeeeeseeeeeeeeeeeeeeeeeas 7 F Exo FITC stain removal and exosome elution 006 10 G List of Components for Flow FACS Kits ccccccceeeeeeees 11 H Additional Materials ReEQuired cccsssceeeeeeeeeeeeeeeeeeeeeeees 11 Exo Flow96 and 32 Exosome IP Kits csesseeeeeeseeeeeees 11 ds Related PrOGUGIS aanne cnir n ieee Matos enteewicns 14 K Shipping and Storage Conditions for Kit ceeeeeeeeeees 14 Il Frequently Asked Questions ccccssseessseeseeeeeeeeeeeeeeeanaaees 14 LIT FICICIGNGCCS aiestiacate arenen aien 14 IM Technical Suppor wzssivececsteicatieent caves ate eee 16 VII Licensing and Warranty information ccc cscceeseeeeeenees 16 Introductio
3. This allows for a more general and inexpensive exosome FACS approach for captured exosome purification The other key feature is that the Exo FITC stain is also reversible Once the captured exosomes on the Exo Flow beads are flow sorted on FITC positive gating the Exo FITC stain can then be washed away and exosomes eluted off of the beads with the Exosome Elution Buffer wash buffer supplied in all kits Add the Exosome Elution Buffer to the exosome bead mixture to 300 400 500 600 simultaneously remove the Exo FITC stain and liberate the intact Wavelength nm exosomes from the beads Then simply place the magnetic beads back on the magnetic stand the eluted and FACS purified exosomes will be in the supernatant The exosome are intact and ready for downstream analysis and functional studies Exo FITC excitation 494nm 7 Exo FITC emission 518 nm Fluorescence Intensity Ww IMPORTANT NOTE If you want to double stain your captured exosomes with a conjugated secondary antibody and the Exo FITC stain you MUST FIRST stain the exosomes on the beads with your conjugated secondary antibody wash then stain with the Exo FITC stain D Exo Flow FACS Protocols To ensure the best success of your flow exometry experiments please follow the protocols exactly as stated below DO NOT vortex the beads when exosomes are bound this will shear them and break them apart leading to no flow sorting positive results Most steps can b
4. 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 Rd Mountain View CA 94043 SBI Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mails General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com Vil Licensing and Warranty information Limited Use License Use of the Exo Flow Kits e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return 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 The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions e 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 e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Purchase of the product does
5. 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 for 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 credit 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 credit 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 Page 16 ver 7 01142014 www systembio com
6. A 5 is 2 06408 aw E E Exosomes O 15E6 08 5 E ET a bed z Add Exosome 5 1 06408 gt Elution Buffer hen T g a E E 5 06407 a 2 Q S O 0E 00 cc Ww 2 3 4006 a w O w Control Eluted Size nen Page 10 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx G List of Components for Flow FACS kits The Exo Flow kits contain enough reagents to perform up to 10 FACS flow exometry experiments Item Amount Streptavidin Magnetic Beads 9 1 um 200 uL at 10 mg mL Biotinylated capture antibody 100 ng uL 100 uL in PBS multiple varieties H Bead Wash buffer Exosome Stain Buffer Exo FITC Universal exosome stain 100 uL Exosome Elution Buffer Magnetic stand optional cat EXOFLOW 700A 1 1 Stand H Additional Materials Required 1 ExoQuick and or ExoQuick TC to concentrate exosome prior to antibody bead capture 2 Magnetic stand or purchase SBI s Multifunctional stand cat EXOFLOW700A 1 3 FACS instrument for flow cytometry 4 Sterile 1 5 mL sample tubes I Exo Flow96 and 32 Exosome IP Kits The Exo Flow96 and 32 IP kits enable the high throughput plate based immunopurification of exosomes directly from serum or from concentrated exosomes from media concentrated with ExoQuick TC or ultracentrifugation The magnetic beads are provided pre coupled with either CD9 CD63 or CD81 antibodies for selective capture of exosomes from either 96 or 32 sample
7. EXOQ20A 1 solution 20 ml adios ExoQuick TC for Tissue Culture Media 10 EXOTC10A 1 and Urine 10 ml reactions ExoQuick TC for Tissue Culture Media and Urine 50 ml eine EXOTC50A 1 Exosome isolation protocol Combine your biofluid sample containing exosomes with ExoQuick or ExoQuick TC using the guidelines shown in the Table below Mix the ExoQuick precipitation reagent with the biofluid sample by inversion and place at 4 C for 30 minutes to overnight then recover the exosomes in a pellet with a low speed spin Please refer to the ExoQuick or ExoQuick TC User manuals for more details Recommended amounts of exosomes provided in Table Resuspend Volume to exosome use in pellet Exo Flow Sample ExoQuick Biofluid volume TC volume Spinal fluid 500 uL PBS 100 uL rxn Culture media 500 uL PBS 100 pL rxn 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Resuspend Volume to ExoQuick exosome use in Exo pellet Flow 250 uL 100 uL rxn Sample Bioflui iofluid volume Ascites fluid 500 uL 120 uL 100 uL rxn Amount of exosomes to use The number of exosomes in a given biofluid will vary depending upon the sample itself There are abundant levels of exosome in serum less in cell culture medium and urine Use the guidelines in the Tables above as a starting point You can typically add about 100 200 ug protein of isolated intact exosomes per Exo Flow reaction Ex
8. e performed at ambient room temperature unless otherwise indicated We highly recommend using concentrated exosomes using SBI s ExoQuick or ExoQuick TC Exosomes isolated through centrifugation for concentration may be used as well Materials 10 reaction kits e Streptavidin Magnetic Beads 9 1 um 400 XL at 10 mg mL 1 6x10 7 beads mL 1 6x10 6 beads mg We will use 0 4 mg per reaction 6 4x10 6 beads per tube Beads will have an orange pumpkin coloring making them Page 4 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx easy to track during the washings e Biotinylated capture antibody 100 ng uL in PBS 100 uL provided examples CD9 Rab5b HLA G depending upon specific kit e Magnetic stand optional cat EXOFLOW700A 1 e Bead Wash buffer 60 mL e Exosome Stain Buffer 3 mL e Exo FITC Universal exosome stain 100 ul provided e Exosome Elution Buffer 3 mL provided ExoQuick and ExoQuick TC are not provided in the Exo Flow kits and can be purchased separately The following ExoQuick products are recommended for exosome concentration prior to Exo Flow purification Description Size Catalog ExoQuick Serum exosome precipitation 75 EXOQS5A 1 solution 5 ml reactions ExoQuick Plasma prep and Exosome precipitation kit 5 ml ExoQuick plus EXOQ5TM 1 Thrombin ae Thrombin Plasma prep for Exosome 100 TMEXO 1 precipitation reactions ExoQuick Serum exosome precipitation
9. exosomes from media samples isolated by ExoQuick TC or ultracentrifugation or directly load serum samples to each bead well for a total volume of 70 ul into the 96 well plate provided Incubate on a shaker at room temperature overnight for exosome capture Place samples on the Exo FlowMag96 magnetic 96 well rack for 2 minutes You should hear a subtle click when the plate is properly aligned with the magnetic plate Keep the clear plate on the magnetic rack and then decant the supernatant Make sure to not disturb the magnetic bead pellets at the bottom of the wells Exosome Bead Capture Photo Exosomes 293 media Serum direct Capture Ab CD9 CD63 CD81 CD9 CD63 CD81 Exosomes on Exosomes on magnetic beads magnetic beads Remove the 96 well plate from the magnetic rack and gently add 50 ul of 1X Wash buffer made fresh from the 20X stock provided Mix by pipetting up and down 3 times and incubate on a shaker at room temperature for 5 minutes Repeat steps 4 6 for a total of 2 washes Remove any residual Wash buffer after decanting and then add 100 ul Exosome Elution Buffer to each well of the 96 well plate Incubate on a shaker at room temperature for 1 hour Place samples plate on the magnetic rack for 2 minutes Carefully remove the supernatant containing eluted intact exosomes and transfer to a fresh tube on ice Make sure not to disturb the magnetic bead pellets Discard the beads afterwards The immunopurified exos
10. for a total volume of 500 uL 16 Incubate on a rotating rack at 4 C overnight for capture Page 6 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx 17 Place samples on magnetic stand for 2 minutes 18 Carefully remove the supernatant after 2 minutes Make sure to not disturb the magnetic bead pellets 19 Remove samples from magnetic stand and add 500 uL Bead Wash buffer Invert a 2 3 times DO NOT VORTEX Flick tubes to mix 20 Place samples on magnetic stand and repeat steps 3 5 for a total of 2 washes Exosome staining 21 Add 240 uL of Exosome Stain Buffer and 10 uL of Exo FITC exosome stain for a final volume of 250 uL per sample 22 Place tubes on ice for 2 hours Flick the tube every 30 minutes to gently mix 23 Place samples on magnetic stand for 2 minutes 24 Carefully remove the supernatant after 2 minutes Make sure to not disturb the magnetic bead pellets 25 Remove samples from magnetic stand and add 500 uL Bead Wash buffer Invert a few times DO NOT VORTEX Flick tubes to mix 26 Place samples on magnetic stand and repeat steps 23 25 for a total of 3 washes 27 Resuspend samples in 300 uL Bead Wash buffer for flow cytometry 28 DO NOT VORTEX prior to loading into FACS instrument Exosome Stain removal and elution if desired 29 Flow sort stained exosome bead complexes as desired 30 Place the stained exosome bead complexes on the magnetic stand for 2 minutes 31 Remo
11. ions and washes can be achieved The Exo Flow kit system also offers a multifunctional magnetic stand as an accessory option catalog EXOFLOW 700A 1 These have been validated to work very well with the Exo Flow beads and can accommodate 12 standard 1 5 mL eppendorf tubes or the stand can be flipped and then used for two standard 15 mL or 50 mL conical tube exosome separations 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Multifunctional Magnetic Separation Stand 50 mLN Magnetic Beads on Stand 15 mL L Tubes Twelve 1 5 mL tubes Beads adhere to side of tube C The Exo FITC Universal and Reversible Stain Once the desired exosomes have been captured and stabilized on the Exo Flow beads you will need to stain these exosomes with a fluorescent moiety to perform the FACS One method is to use a second antibody conjugated to PE APC ALEXAs or FITC to accomplish this goal These typically offer low staining ability and can be costly for multiple exosome sample purifications SBI has invented a novel method to universally stain captured exosomes on the beads The technology takes advantage of the findings that most exosome surface proteins have modifications ex glycosylations carbohydrate additions etc The Exo FITC stain supplied in all kits features FITC fluorescent molecules conjugated to a protein known to universally bind these types of protein modifications
12. lysis and sorting The Exo Flow kits are modular thus you can select from various pre validated capture antibody kits or utilize your own biotinylated capture antibody 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual corresponding to the exosome surface marker specific for the exosomes of interest in your model system SBI has thoroughly tested a variety of capture antibodies that work quite well to flow sort exosomes from either serum or cell culture samples Flow Exometry SBI has developed a simple one step method for precipitation of all exosomes from biofluids using polymer formulations The ExoQuick reagent can be used to isolate exosomes from serum plasma and tumor ascites fluids Exosomes from more dilute larger volume fluids like cell culture media using cerebral spinal fluid can be efficiently isolated using SBI ExoQuick TC While ExoQuick isolation of exosomes is rapid and easy this method will isolate every exosome from your biofluid sample The Exo Flow kits are designed to enable the selective capture and flow sorting to purify distinct subpopulations of exosomes based on a particular surface marker You will first enrich for all exosomes using ExoQuick serum plasma ascites samples or ExoQuick TC cell media urine spinal fluid The isolated exosomes are then resuspended and bound to the magnetic beads for specific capture and subsequent flow exometry The E
13. n A Exosome Overview Exosomes are 60 180 nm membrane vesicles secreted by most cell types in vivo and in vitro These microvesicles are produced by the inward budding of multivesicular bodies MVBs and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane Exosomes are extracellular nanoshuttle organelles that facilitate communication between cells and organs Exosomes are found in blood urine amniotic fluid breast milk malignant ascites fluids and contain distinct subsets of RNAs and proteins depending upon the cell type from which they are secreted making them useful for biomarker discovery SBI has engineered tools and next generation sequencing services to accelerate the study of exosomes exosome protein and RNA biomarkers The Exo Flow kits have two major applications 1 Purify exosome with specific surface markers for FACS analytics and purification and 2 high throughput immunopurification IP of exosomes from media serum and other biofluids in 96 well formats The Exo Flow kits for FACS These kits are for the selective capture and flow sorting to purify distinct subpopulations of exosomes based on a particular surface marker You will first enrich for all exosomes using ExoQuick serum plasma ascites samples or ExoQuick TC cell media urine spinal fluid The isolated exosomes are then resuspended and bound to the magnetic beads for specific capture and subsequent FACS ana
14. ng J Immunol 2013 May 17 Epub ahead of print Narayanan A lordanskiy S Das R Van Duyne R Santos S Jaworski E Guendel Sampey G Gerhart E Iglesias Ussel M Popratiloff A Hakami R Kehn Hall K Young M Subra C Gilbert C Bailey C Romerio F Kashanchi F Exosomes derived from HIV 1 infected cells contain TAR RNA J Biol Chem 2013 May 9 Epub ahead of print Carlos Salomon Luis Sobrevia Keith Ashman Sebastian E Illanes Murray D Mitchell and Gregory E Rice The Role of Placental Exosomes in Gestational Diabetes Mellitus Gestational Diabetes Causes Diagnosis and Treatment Dr Luis Sobrevia Ed ISBN 978 953 51 1077 4 InTech DOI 10 5772 55298 Skrzypek K Tertil M Golda S Ciesla M Weglarczyk K Collet G Guichard A Kozakowska M Boczkowski J Was H Gil T Kuzdzal J Muchova L Vitek L Loboda A Jozkowicz A Kieda C Dulak J Interplay between heme oxygenase 1 and miR 378 affects non small cell lung carcinoma growth vascularization and metastasis Antioxid Redox Signal 2013 Apr 25 Epub ahead of print Haney Mu Zhao Y Harrison EB Mahajan V Ahmed S et al Specific Transfection of Inflamed Brain by Macrophages A New Therapeutic Strategy for Neurodegenerative Diseases PLoS ONE 8 4 e61852 doi 10 1371 journal pone 0061852 Hu G Gong AY Roth AL Huang BQ Ward HD Zhu G Larusso NF Hanson ND Chen XM Release of Luminal Exosomes Contributes to TLR4 Mediated Epithelial Antimicrobial Defe
15. nse PLoS Pathog 2013 Apr 9 4 e1003261 Danny A Kanhai Frank L J Visseren Yolanda van der Graaf Arjan H Schoneveld Louise M Catanzariti Leo Timmers L Jaap Kappelle Cuno S P M Uiterwaal Sai Kiang Lim Siu Kwan Sze Gerard Pasterkamp Dominique P V de Kleijn on behalf of the SMART Study Group Microvesicle protein levels are associated with increased risk for future vascular events and mortality in patients with clinically manifest vascular disease International Journal of Cardiology ISSN 0167 5273 10 1016 j ijjcard 2013 01 231 PDF Katakowski M Buller B Zheng X Lu Y Rogers T Osobamiro O Shu W Jiang F Chopp M Exosomes from marrow stromal cells expressing miR 146b inhibit glioma growth Cancer Lett 2013 Feb 15 pii SO304 3835 13 00131 6 doi 10 1016 j canlet 2013 02 019 Choi DS Kim DK Kim YK Gho YS Proteomics transcriptomics and lipidomics of exosomes and ectosomes Proteomics 2013 Feb 11 dol 10 1002 omic 201200329 Epub ahead of print Munch EM Harris RA Mohammad M Benham AL Pejerrey SM Showalter L Hu M Shope CD Maningat PD Gunaratne PH Haymond M Aagaard K Transcriptome Profiling of microRNA by Next Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk PLoS One 2013 8 2 e50564 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual IV Technical Support For more information about
16. o Flow FACS Magnetic bead preparation 1 Vortex bead slurry briefly and then pipette 40 uL of bead slurry solution into a 1 5 mL tube per sample 2 Place samples on magnetic stand for 2 minutes 3 Carefully remove the supernatant after 2 minutes Make sure to not disturb the magnetic bead pellets 4 Remove samples from magnetic stand and add 500 uL of Bead Wash buffer Invert a few times 5 Place samples on magnetic stand and repeat steps 2 4 for a total of 2 washes 6 Remove all liquid so only beads are on the side of the tube Binding of capture antibody to beads 7 Remove tubes from magnetic stand and add 10 uL of biotinylated capture antibody ie CD9 CD63 per sample using the pipette tip to move the beads to the bottom of the tube mix by pipetting up and down three times 8 Place tubes on ice for 2 hours Flick the tube every 30 minutes to gently mix 9 Add 200 uL Bead Wash buffer and flick to mix 10 Place samples on magnetic stand for 2 minutes 11 Carefully remove the supernatant after 2 minutes Make sure to not disturb the magnetic bead pellets 12 Remove samples from magnetic stand and add 500 uL Bead Wash buffer Invert a few times DO NOT VORTEX Flick tubes to mix 13 Place samples on magnetic stand and repeat steps 10 12 for a total of 3 washes 14 Suspend capture antibody beads with 400 uL Bead Wash buffer per sample Exosome capture 15 Add 100 uL of concentrated isolated exosomes to each bead sample
17. omes are stable at 4 C for several days or can be frozen at 80 C for long term storage Example data of exosome recoveries for intact exosomes and the number of exosome particles can be seen in the Figure below Page 12 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx Exosome concentrations recovered using the Exo Flow IP kits were measured by NanoSight analysis below The data below demonstrate the exosome recoveries with sizes centered around 88 nm in diameter The HEK 293 media exosomes were purified from 20 ml media and concentrated using ExoQuick TC and resuspended in 1 mL PBS Then 50 ul of the concentrated media exosomes were added per well for the immunopurifications The serum exosomes were isolated from 50 ul of human serum added directly to the bead capture mixture NanoSight Exosome Analysis 7 20E 09 293 Exosomes 6 20E 09 Number Exosome Particles 1 20E 09 2 00E 08 5 20E 09 4 4 20E 09 4 3 20E 09 2 20E 09 CD81 capture 88nm ___ 293 exosomes CD81 capture Serum exosomes CD9 capture Serum exosomes CD63 capture Serum Exosomes CD9 capture Serum Exosomes CD63 capture 0 50 100 150 200 Particle Diameter nm Optional Western blotting Protocol a A To the eluted exosome supernatant containing add 1 ul of the clearing reagent this dissolves the elution buffer away from the exosomes Invert it a few
18. osome pellet was resuspended in 500 ul 1x PBS The amount of exosome particles were added in two fold dilutions starting at 50 ul and then captured using the biotinylated CD9 antibody and Exo Flow beads The FITC flow cytometric intensities are then plotted versus the number of exosome particles input into the flow reaction The Exo Flow system has the capacity to capture flow and detect BILLIONS of exosomes CD9 Beads Exo FITC Serum Exosome Capture Titration N Pd 2 FITC fluorescence 0 1 00E 10 2 00E 10 3 00E 10 4 00E 10 5 00E 10 Exosomes 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual F Exo FITC stain removal and exosome elution Once you have flow sorted the captured exosomes of interest the Exo FITC stain can be easily removed and intact exosomes eluted safely This can enable you to then perform downstream analysis and functional studies The data below show the efficient Exo FITC stain removal and exosome elution The flow sort results from CD9 captured exosomes stained with Exo FITC and then eluted with the Exosome Elution Buffer The entire bead population returns to pre stain FITC intensities and NanoSight studies verify the recovery of intact exosomes O09 Beads Exo FITC CDS Beads Exo FITC ince NO Exosenpes PLUS Exosonves Exosome Elution Buffer Fac H i H i i F F Mw w wo w w ow w a o lO a w w w wt FUT 7 255408 Wi x CD9 Exo ELIS
19. oupled with Exo FITC staining are shown below The data are graphed showing forward scatter versus FITC intensity The first panel depicts beads with no exosomes then with exosomes The degree of flow separation is shown on the right side for each capture set CDO Beads Exo FITC NO Exosomes 0 5 FITC Positive om 0 a i HF Vi ui CD31 Beads Exo FITC NO Exosomes 0 3 FITC Positive CD63 Beads Exo FITC NO Exosomes 0 4 FITC Positive o nan CD81 Beads Exo FITC NO Exosomes 1 4 FITC Positive aa aa LURID ED9 Beads Exo FITC PLUS Exosomes CD31 Beads Exo FITC PLUS Exosomes 99 9 FITC a Positive CD63 Beads Exo FITC PLUS Exosomes 99 4 FITC Positive CD81 Beads Exo FITC PLUS Exosomes 100 FITC Positive CD9 Beads Exo FITC PLUS Exosomes PLUS Exosomes CD63 Beads Exo FITC US Exosomes CD81 Beads Exo FITC PLUS Exosomes ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx RabSb Beads Exo FITC Rab5b Beads Exo FITC Rab5b Beads Exo FITC NO Exosomes PLUS Exosomes PLUS Exosomes 0 76 FiTc 99 9 FITC Positive Positive HLA G Beads Exo FITC HLA G Beads Exo FITC HLA G Beads Exo FITC NO Exosomes PLUS Exosomes PLUS Exosomes 1 1 FITC 99 8 FITC Positive a Positive Exo Flow FACS exosome binding capacity data Human serum exosomes were isolated from 250 ul serum using ExoQuick The ex
20. s simultaneously The Exo Flow96 kits contain all of the reagents for exosome IP purifications from 96 samples and the Exo Flow32 kit has all of the reagents for 32 samples Exosomes in serum or plasma samples can be immunopurified directly by adding to the IP magnetic beads We recommend concentrating exosomes first from media urine and CSF before immunopurifcation Choose from exosome IP antibodies for CD9 CD63 or CD81 surface markers The exosome are immunopurified and eluted intact allowing for downstream functional studies as well as for exoRNA and protein biomarker analyses Materials provided e 96 well clear plate 8 well strips and covers e Capture Antibody pre coupled with magnetic beads e Wash buffer 20X stock e Exosome Elution Buffer e Clearing reagent to dissolve elution reagent away from exosomes e Exo FlowMag96 magnetic 96 well rack sold separately 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual Protocol for ExoFlow96 or 32 Plate Magnet alone Clear Plate on Magnet 10 11 12 ee Ww gt er a ap ted D on _ te in he en ian Se ed SA A ee ote i Lam Loe Lge tne tre sp tae Neh sch ot om Vhs pia La is et at pt k s tix m int sO Ee 1 pe tye pe re ee D D Pa d J Pa Add 20 ul of antibody coupled with magnetic beads to each well of the plate provided per capture reaction Add 50 uL of concentrated isolated
21. termined experimentally for each antibody tried We recommend starting with at least 50 ug secondary antibody The Exo FITC stain can serve as a positive control for the presence of exosomes during your FACS analysis Q What are the species cross reactivities of each of the Exo Flow antibodies All of the Exo Flow capture antibodies have been optimized for human exosome capture Due to the high protein sequence conservation of many of these exosome surface markers cross species reactivity is likely but SBI has not tested all mammalian species yet The Rab5b biotinylated antibody has the most cross species capture reactivity Human Mouse Rat Chicken Pig Cow Horse Rabbit Sheep and can be used to develop your exosome flow sorting experiments References Huang X Yuan T Tschannen M Sun Z Jacob H Du M Liang M Dittmar RL Liu Y Liang M Kohli M Thibodeau SN Boardman L Wang L Characterization of human plasma derived exosomal RNAs by deep sequencing BMC Genomics 2013 May 10 14 319 Kurbegovic A Trudel M Progressive development of polycystic kidney disease in the mouse model expressing Pkd1 extracellular domain Hum Mol Genet 2013 Jun 15 22 12 2361 2375 Page 14 ver 7 01142014 www systembio com Exo Flow Exosome Capture Kits Cat EXOFLOWxxx Bhattarai N McLinden JH Xiang J Landay AL Chivero ET Stapleton JT GB Virus C Particles Inhibit T Cell Activation via Envelope E2 Protein Mediated Inhibition of TCR Signali
22. the membrane on a plastic surface for imaging Detect signals with chemi luminescence and expose the membrane for 5 45 seconds to image signals crtn nro J Related Products SBI offers a number of exosome research products Review them here htip www systembio com exosomes e ExoQuick exosome isolation reagents e Exo FBS exosome depleted media supplement e Detect and quantitate exosomes with Antibodies and ELISAs e Purify exosome RNA and profile by qPCR with SeraMir e Discover novel exoRNA biomarkers with Next Gen sequencing services K Shipping and Storage Conditions for Kit The Exo Flow kits are shipped on blue ice and should be stored at 4 C Avoid freeze thawing the reagents Shelf life of the product is 1 year after receipt if stored in 4 C Frequently Asked Questions Q How many exosomes should I start with before flow sorting The biofluid type and cell culture media source will determine just how many exosomes are present The best way is to first isolate and concentrate the exosomes of interest Resuspend the isolated exosomes in a minimal volume 10 of starting volume with 1X PBS Take a protein concentration measurement and use approximately 50 ug to 200 ug exosome protein input per Exo Flow capture experiment Q If want to detect the captured exosomes using a fluorescently conjugated secondary antibody how much should I use Every antibody is different and will have varying degrees of affinity This is best de
23. times to mix it well Incubate at 37 C for 30 minutes the sample is now ready for Westerns Measure the protein concentration of the samples Load 1 5 ug protein sample per well Add 10 ul of 5x loading dye to the sample in a total of 50 ul and boil for 5 minutes at 95 C Perform standard SDS PAGE electrophoresis and Western transfer onto nitrocellulose membranes Block with 5 dry milk in Tris Buffered Saline 0 05 Tween TBS T for 1 hour or Blocking buffer for 1 hour Incubate blot overnight at 4 C with exosome specific primary antibody eg Anti CD63 at 1 1 000 dilution 5 dry milk in TBS T Drain the antibody solution Wash with TBS T for 3X 5 minutes Drain the wash solution Western Blot Analysis Exosomes 293media Serum direct Capture Ab CD63 CD9 CD63 CD9 Detection Ab CD63 gt 50 kD Detection Ab CD9 gt 25 kD Prepare exosome validated secondary antibody at 1 20 000 dilution 5 dry milk in TBS T Incubate one hour at room temperature with exosome validated secondary antibody at 1 20 000 dilution 5 dry milk in TBS T Wash with TBS T for 3X 5 minutes Drain the wash solution Prepare detection solution 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Add 5 ml developing solution to the membrane Incubate at room temperature for 2 minutes Take the membrane out of the dish drain excess detecting solution using kimwipe Place
24. ve buffer and add 300 ul Exosome Elution Buffer Invert a few times DO NOT VORTEX Flick tubes to mix 32 Incubate on a rotating rack or shaker at 25 C for 2 hours 33 Place samples on magnetic stand for 2 minutes 34 Carefully remove the supernatant containing your eluted exosomes and transfer to a fresh tube Make sure to not disturb the magnetic bead pellets Discard beads after use E Sample Exo Flow FACS data The data below were generated on a BD LSR II instrument and are meant as examples of how to set the gate settings and perform data analysis using FlowJo software Information on FlowJo software can be viewed online http www flowjo com Exo Flow software gate settings The forward and side scatter data for the 9 1 um Exo Flow beads are shown below for samples containing no captured exosomes stained with Exo FITC left panel and then data for serum CD9 captured exosomes stained with 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Exo FITC Set the gate primarily on the majority bead singlets outlined in a black oval red arrow pointing to the gate setting prior to full flow analysis CD9 Beads Exo FITC NO Exosomes CD9 Beads Exo FITC PLUS Exosomes Gate on Gate on Singlets Singlets 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC A FSC A Exo Flow exosome flow exometry example data Bead flow separation data for the various capture antibodies c
25. xo Flow kits for High throughput IP The Exo Flow96 and 32 IP kits enable the high throughput plate based immunopurification of exosomes directly from serum or from concentrated exosomes from media with ExoQuick TC or ultracentrifugation The magnetic beads are provided pre coupled with either CD9 CD63 or CD81 for selective capture of distinct subsets of exosomes from either 96 or 32 samples simultaneously Why is this important Exosomes found in biofluids can originate from any tissue or cell type and end up in the serum fraction If you are interested in just the immune related exosomes you would need to capture them using a specific exosome surface marker like HLA G same goes for tumor derived exosome populations The most efficient method for exosome subpopulation purification is using the Exo Flow kits B Magnetic Beads and Exosome Surface Markers SBI has developed a magnetic streptavidin 9 1 um Exo Flow bead system Fyo Fl oy chart ee with ultra high exosome binding capacity The 9 1 um diameter of the Reads pepe A este pcan beads enables more exosome capture per volume added when compared Biotin conjugated Capture Antibody to 4 um Dynabeads This is significant in that some exosome A m V corny oime subpopulations that are desired may only be present in very low numbers A r o The increased surface area enables the more efficient capture of these _m rare exosomes Bigger is better Outer core hydrophilic polymer surface
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