Exo-GLOW™ Exosome Labeling Kits
Contents
1. 500 ug per labeling reaction Each Exo Glow reaction rxn equates to labeling 1 5 x 1048 exosomes Protocol for labeling exosomes 1 Add 50 ul 10x Exo Red or Exo Green to 500 uL volume of resuspended exosome suspension in 1x PBS in a 1 5 ml eppendorf tube 2 Mix it well by flicking inversion Do not vortex 3 Incubate the exosome solution in 37 C for 10 minutes rotation not necessary 4 To stop labeling reaction add 100 ul of the ExoQuick TC reagent to the labeled exosome sample suspension and mix by inverting 6 times 5 Place the labeled exosome sample on ice or at 4 C for 30 minutes 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual 6 7 Centrifuge the sample for 3 minutes at 14 000 rpm ina microfuge Remove the supernatant with excess label and resuspend the labeled exosome pellet in 500 ul 1x PBS The labeled exosomes are ready to be monitored for tracking Adding Exo Glow exosomes to cells 1 Add at least 100 ul of labeled exosomes to approximately 1x10 5 cells per well in a 6 well culture plate as an example You can scale this ratio up or down depending upon your experimental requirements 2 Incubate for 2 to 24 hours visualize with fluorescent microscopy using the following excitation emission guidelines below Exosome Label Excitation Emission Filter setting Exo Red 460 nm 650 nm red Typical RFP filter set Exo2. For additional information or technical assistance please call or email us at Page 12 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Phone 650 968 2200 SBI 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 Glow Kits i 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 8
3. Green 494 nm 521 nm green Typical GFP filter set NOTE ExoQuick and ExoQuick TC for exosome isolation purposes are not provided in the Exo Glow kits and can be purchased separately The following ExoQuick products are recommended for exosome concentration prior to Exo Flow purification Page 4 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 Description Size Catalog ExoQuick Serum exosome precipitation 75 l EXOQ5A 1 solution 5 ml reactions ExoQuick Plasma prep and Exosome 75 precipitation kit 5 ml ExoQuick plus EXOQ5TM 1 i reactions Thrombin Thrombin Plasma prep for Exosome 100 l TMEXO 1 precipitation reactions ExoQuick Serum exosome precipitation 300 l EXOQ20A 1 solution 20 ml reactions ExoQuick TC for Tissue Culture Media 10 _ EXOTC10A 1 and Urine 10 ml reactions ExoQuick TC for Tissue Culture Media 50 and Urine 50 ml reactions EXOTCS0A I Exosome isolation protocols using ExoQuick reagents 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 provi
4. 88 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual 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 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 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 impl
5. SBI System Biosciences Exo GLOW Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 User Manual Store Kits at 20 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 Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 Contents le INTODUCHONicdivs edean ce baeniaveecind a aE 1 A ExOSOME OvVerVie W dorsier ioanen eraa a Ra aaa aani 1 B The Exo Glow kits for labeling EXOSOMeS sessseeeeese eenen 2 Cy EXO Glow Protocols y ortaca aeaaaee n araea Eas 3 D Sample Exo Glow IP bead data ccccecssceeeeeeeeeeeeseeeeeeee 6 E Exo Glow exosomes added to target cells c eeeeeeeee 8 F How the exosome labels work ccceeseeeeeceeeeeeeeeeeeneeeeee 9 G Additional Materials Required ccccceeeeeeeeeeeeeseeeeeneeees 10 H Related Products in t2 c ccl aie ee 10 l Shipping and Storage Conditions for Kits cccceeereees 10 Il References vac ct aia e alate iat es 11 lil Technical Support cceeccceceeeceeeeeeeeeeeeeeeeseaeeeeaeseeneeeeaes 12 VII Licensing and Warranty information c cccceeeeeeeees 13 l Introduction A Exosome Overview Exosomes are 60 180 nm membrane vesicles secreted by most cell types in vivo and in vitro These microvesicles are produ
6. arcinoma growth vascularization and metastasis Antioxid Redox Signal 2013 Apr 25 Epub ahead of print Haney MJ 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 Defense PLoS Pathog 2013 Apr 9 4 e1003261 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 doi 10 1002 pmic 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 lll Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site hitp www systembio com
7. arget cells via the exosomes using fluorescent microscopy The Exo Green stain is based on Carboxyfluorescein succinimidyl diacetate ester CFSE chemistry CFSE is also membrane permeable When CFSE enters exosomes it is hydrolyzed by exosome esterases known to be within exosomes that remove the diacetate residues Savina A et al 2002 This activates the CFSE to fluoresce green and is then coupled to the amino ends of proteins This approach allows researchers to track exoProtein transfer into target cells using fluorescent microscopy Fluorescently label exosome internal RNA Red or internal exosome proteins Green for tracking Page 2 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 C Exo Glow Protocols Materials provided e Exo Red 10x in DMSO 1 ml per kit e Exo Green 10x in water 1 ml per kit e ExoQuick TC 2 ml per kit Description Cat Size Exo Red exosome RNA fluorescent label EXOR100A 1 20 rxns Exo Green exosome protein fluorescent label EXOG200A 1 20 rxns Exo Red Exo Green exosome cargo fluorescent label Combo EXOC300A 1 20 rxns Pack 10 rxns of each label Exosome sample preparation Isolate exosomes from serum or media isolated by ExoQuick ExoQuick TC or ultracentrifugation methods resuspend the exosome pellet in 500 ul of 1x PBS The recommended exosome protein input should be approximately 100
8. ced 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 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual 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 B The Exo Glow kits for labeling exosomes The Exo Glow kits allow researchers to fluorescently label isolated exosomes to track cellular interaction and uptake You will first enrich for all exosomes using ExoQuick serum plasma ascites samples or ExoQuick TC cell media urine spinal fluid or immunopurify specific exosome subpopulations using SBI s Exo Flow IP kits The isolated exosomes are then resuspended and incubated with either the 10x Exo Red or 10x Exo Green labeling stain The Exo Red stain is based on an Acridine Orange AO chemistry AO is membrane permeable and fluorescently labels single stranded RNAs inside of exosomes These Red exoRNAs can then be monitored for delivery into t
9. ded in Table Resuspend Volume to exosome use in pellet Exo Flow Sample ExoQuick Bioflui giud volume TC volume Spinal fluid 500 uL PBS 100 uL rxn Culture media 500 uL PBS 100 uL 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 Ascites fluid 500 pL 120 pL aoe 100 pLirxn 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 100 500 ug protein of isolated exosomes per Exo Glow reaction Sample Biofluid volume D Sample Exo Glow IP bead data Exosomes were first isolated from the tissue culture medium from HEK293 cells grown in Exo FBS exosome depleted media supplement with standard DMEM The cells were grown to 80 90 confluency in a 10 cm cell culture dish The secreted exosomes were isolated as stated in the protocol for ExoQuick TC above The exosome pellet was resuspended in 1 ml 1x PBS and contained an exosome protein content of 1 ug ul About 500 ul of this exosome suspension was labeled with 50 ul of either 10x Exo Red or Exo Green for 10 minutes at 37 C The exosomes were re isolated using the addition of 100 ul ExoQuick TC and precipitation at 5 C fo
10. 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 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 Signaling 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 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual small cell lung c
11. em Biosciences SBI User Manual How the Exo Green label works Label internal exosome proteins green Exo Green CFSE CFSE Proteins Esterase Coupling to hydrolysis amino termini G Additional Materials Required 1 ExoQuick and or ExoQuick TC to isolate exosomes prior to labeling 2 Standard 1x PBS 3 Sterile 1 5 mL sample tubes H Related Products SBI offers a number of exosome research products Review them here http Awww systembio com exosomes 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 l Shipping and Storage Conditions for Kits The Exo Glow kits are shipped in brown light protection tubes on dry ice and should be stored at 20 C Avoid freeze thawing the reagents Shelf life of the product is 1 year after receipt if stored in 4 Page 10 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 ll References Savina A Vidal M Colombo MI The exosome pathway in K562 cells is regulated by Rab11 J Cell Sci 2002 Jun 15 115 Pt 12 2505 15 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
12. ied 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 2014 System Biosciences SBI All Rights Reserved Page 14 ver 7 071614 www systembio com
13. labeled exosomes on cells 2 hours later track RNA delivery Page 8 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 F How the exosome labels work The Exo Red exosome label is based on Acridine Orange chemistry and is a nucleic acid selective fluorescent cationic dye It is cell permeable and interacts with DNA by intercalation and RNA by electrostatic attractions When bound to DNA Exo Red is very similar spectrally to fluorescein with an excitation maximum at 502 nm and an emission maximum at 525 nm green When Exo Red associates with RNA the excitation maximum shifts to 460 nm blue and the emission maximum shifts to 650 nm red This allows for the labeling of internal exosome RNAs and the ability to monitor RNA delivery to cells via exosomes Label internal exosome RNAs red OQO g SS P o Binds single stranded exoRNA fluoresces red The Exo Green stain is based on Carboxyfluorescein succinimidyl diacetate ester CFSE chemistry CFSE is also membrane permeable When CFSE enters exosomes it is hydrolyzed by esterases known to be within exosomes that remove the diacetate residues This activates the CFSE to fluoresce green and is then coupled to the amino ends of proteins This approach allows researchers to track exosome Protein transfer into target cells using fluorescent microscopy 888 266 5066 Toll Free 650 968 2200 outside US Page 9 Syst
14. r 30 minutes on ice or 4 C The labeled exosome pellets were resuspended in 500 ul 1x PBS CD63 coupled magnetic beads from SBI s Exo Flow IP kit cat EXOFLOW32A CD63 Briefly 50 ul of CD63 magnetic beads were incubated with 100 ul of the labeled exosomes overnight at 4 C on a rotator The following day the bead labeled exosomes were placed on a magnetic plate for 5 minutes and then washed with 100 ul 1x wash buffer once Then 100 ul 1x PBS was added to the IP well and placed on the magnetic rack for 2 minutes to position the beads at Page 6 ver 7 071614 www systembio com Exo Glow Exosome Labeling Kits Cat s EXOR100A 1 EXOG200A 1 EXOC300A 1 the bottom of the wells The beads with captured labeled exosomes were imaged using fluorescent microscopy Exo Red labeled exosomes bound to CD63 beads ER labeled exosomes 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Exo Green labeled exosomes bound to CD63 beads hse gt o o o2 o hi 2 EG labeled exosomes E Exo Glow exosomes added to target cells The same labeled exosomes from E above were added to target HEK 293 cells in culture The cells were plated at about 1x10 5 cells in a standard 6 well culture plate Approximately 100 ul of the labeled exosome suspension was added per 6 well with cells and allowed to interact uptake for the time indicated in the following data figures Exo Red
Download Pdf Manuals
Related Search