XPack™ Exosome Protein Engineering Technology
Contents
1. of XPack reporter proteins on target cells in a 6 well plate format The number of exosomes required in culture dishes of other size can be scaled up or down proportionally to the difference in total cell number relative to one well of a 6 well plate Example HEK293T cells 6 well seeding density 400 000 cells 24 well seeding density 100 000 cells 100 000 400 000 1 4 number of cells 250 ug exosomes x 14 62 5 ug exosomes for use in 24 well plate format VI References Gy rgy B Hung ME Breakefield XO Leonard JN Therapeutic applications of extracellular vesicles clinical promise and open questions Annu Rev Pharmacol Toxicol 2015 55 439 64 doi 888 266 5066 Toll Free 650 968 2200 outside US Page 23 System Biosciences SBI User Manual 10 1146 annurev pharmtox 010814 124630 Epub 2014 Oct 3 PubMed PMID 25292428 van der Meel R Fens MH Vader P van Solinge WW Eniola Adefeso O Schiffelers RM Extracellular vesicles as drug delivery systems lessons from the liposome field J Control Release 2014 Dec 10 195 72 85 doi 10 1016 j jconrel 2014 07 049 Epub 2014 Aug 2 Review PubMed PMID 25094032 Coleman BM Hill AF Extracellular vesicles Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases Semin Cell Dev Biol 2015 Feb 20 pii S1084 9521 15 00034 8 doi 10 1016 j semcdb 2015 02 007 Epub ahead of print Review PubMed PMID 25704308 Yao Y Wei W2. 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 28 ver 1 150316 www systembio com
3. using SBI s PureFection Cat LV750A 1 in a 6 well plate of cells at 70 80 confluency a Mix 5 uL PureFection reagent 2 5 ug XPack Lentivector and 200 uL serum free media in sterile 1 5 mL Eppendorf tube 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 3 4 b Vortex briefly and incubate at room temperature for 15 minutes c Add entire volume to 6 well of cells in a total volume of 2 3 ml media Change media after 24 hours Isolate exosomes in 48 96 hour window post transfection Isolation of XPack Exosomes and Addition to Target Cells 1 Remove cell culture media and place in 15 mL or 50 mL centrifuge tube Add ExoQuick TC at 1 5 the volume of cell culture media Mix by inversion and incubate at 4 C overnight Spin centrifuge tubes at 3 000 x g for 30 minutes at room temperature or 4 C temperature does not affect exosome yield Discard supernatant and resuspend exosome containing pellet in 100 uL PBS Measure exosome yield using A280 on Nanodrop Adjust concentration to 1 ug uL Add exosomes to cell culture dish containing target cells For target cells gt 1 5x10 5 cells in a 6 well plate format 250 ug exosomes is sufficient to visualize efficient delivery of XPack GFP using fluorescence microscopy The number of exosomes required to discern effects in target cells may vary by cell type and by the specific phenotype being assayed therefore optimizati
4. 2 IV XPack Purified EXOSOMES ccceceeeeesteeeeeteteeeeeeneeeenene 22 V Frequently Asked Questions 0 cceeceeeeeeeeeeteeeeteeeeeeeeees 23 VI REICIENCES aione eaea a ET a E S 23 Vik Technical SUpport Areeiras 26 VIII Licensing and Warranty information cc eeeeeeeeeeeeees 27 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual Introduction A XPack Overview Exosomes are nanosized membrane vesicles secreted by most cell types in vivo and in vitro They are produced by the inward budding of multivesicular bodies MVBs and subsequently released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane Exosomes are extracellular nanoshuttles that facilitate communication between cells and organs and are found in various biofluids including blood urine amniotic fluid breast milk malignant ascites fluid and cerebrospinal fluid CSF Exosomes contain distinct subsets of RNAs and proteins depending upon the cell type from which they are secreted making them useful for biomarker discovery Additionally their natural function as cell to cell communication vehicles makes them attractive for use as therapeutic shuttles to deliver biological molecules or drugs to target disease cells SBI has identified a specific peptide sequence that targets a protein to the interior exosomal membrane allowing them to be packaged into exosomes for
5. Proteomics 2014 Apr 11 2 167 77 doi 10 1586 14789450 2014 890894 Epub 2014 Feb 25 PubMed PMID 24564711 Soldevilla B Rodriguez M San Millan C Garcia V Fernandez Peria ez R Gil Calder n B Martin P Garcia Grande A Silva J Bonilla F Dominguez G Tumor derived exosomes are enriched in ANp73 which promotes oncogenic potential in acceptor cells and correlates with patient survival Hum Mol Genet 2014 Jan 15 23 2 467 78 doi 10 1093 hmg ddit437 Epub 2013 Sep 18 PubMed PMID 24067531 Camacho L Guerrero P Marchetti D MicroRNA and protein profiling of brain metastasis competent cell derived exosomes PLoS One 2013 Sep 16 8 9 e73790 doi 10 1371 journal pone 0073790 eCollection 2013 PubMed PMID 24066071 PubMed Central PMCID PMC3774795 Raimondo F Morosi L Corbetta S Chinello C Brambilla P Della Mina P Villa A Albo G Battaglia C Bosari S Magni F Pitto M Differential protein profiling of renal cell carcinoma urinary exosomes Mol Biosyst 2013 Jun 9 6 1220 33 doi 10 1039 c3mb25582d Epub 2013 Mar 19 PubMed PMID 23511837 de Jong OG Verhaar MC Chen Y Vader P Gremmels H Posthuma G Schiffelers RM Gucek M van Balkom BW Cellular stress conditions are reflected in theprotein and RNA content of endothelial cell derived exosomes J Extracell Vesicles 2012 Apr 888 266 5066 Toll Free 650 968 2200 outside US Page 25 System Biosciences SBI User Manual 1631 doi 10 3402 jev v1i0 18396 eColle
6. SBI System Biosciences XPack Exosome Protein Engineering Technology Cat s XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 User Manual Store at 20 C upon arrival 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 XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Contents l troduction scaon a 2 A XPack OVEIVICW cccceeeceeeeeceeeee cee eeeeaeseeneeseeeesaeeneneeeeaas 2 B Uses of XPack TeChHNOlOGY cccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeaees 2 Il XPack Lentivector SySteM cccecccecseeeeseeeeeeeeseteeeeeaeeeeneeees 4 A List of Constructs and Pre Packaged Particles 4 B XPack Transfection Protocol cccceeeeeeeeeeeeeeeeeeeeeeees 5 D XPack Lentiviral Particle Production 0 eceeeeeeeee 8 E Transduction of Pseudoviral Particles into Target Cells 11 F XPack Sample Datta cccecccccseeeecceeeeeseeceeeeessaeeeeneeeeeees 12 G Shipping and Storage Conditions ceceeeeesteeeeenees 18 Fi Related Producted onsi nn a E EAEE 19 III XPack Stable Cell LIn S c ccceeeeeeeeeeeeeeeeeeeeseeeeeeeeeaes 19 A XPack Stable Cell Line Validation Data cee 19 B XPack Stable Cell Growth Conditions ceeeee 21 Shipping and Storage Conditions cccceeeeseeeeeees 2
7. Sun J Chen L Deng X Ma L Hao S Proteomic analysis of exosomes derived from human lymphoma cells Eur J Med Res 2015 Jan 29 20 1 8 PubMed PMID 25631545 PubMed Central PMCID PMC4329659 Yang J Wei F Schafer C Wong DT Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 2014 Nov 14 9 11 e110641 doi 10 1371 journal pone 0110641 eCollection 2014 PubMed PMID 25397880 PubMed Central PMCID PMC4232306 Zhao X Wu Y Duan J Ma Y Shen Z Wei L Cui X Zhang J Xie Y Liu J Quantitative proteomic analysis of exosome protein content changes induced by hepatitis B virus in Huh 7 cells using SILAC labeling and LC MS MS J Proteome Res 2014 Dec 5 13 12 5391 402 doi 10 1021 pr5008703 Epub 2014 Oct 8 PubMed PMID 25265333 Revenfeld AL B k R Nielsen MH Stensballe A Varming K J rgensen M Diagnostic and prognostic potential of extracellular vesicles in peripheral blood Clin Ther 2014 Jun 1 36 6 830 46 doi 10 1016 j clinthera 2014 05 008 PubMed PMID 24952934 Page 24 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Zhang L Wrana JL The emerging role of exosomes in Wnt secretion and transport Curr Opin Genet Dev 2014 Aug 27 14 9 doi 10 1016 j gde 2014 03 006 Epub 2014 May 8 Review PubMed PMID 24791688 Drake RR Kislinger T The proteomics of prostate cancer exosomes Expert Rev
8. age Please follow the recommended guidelines for working with BSL 2 biosafety agents 2 Concentration of Pseudoviral Particles 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual The PEG it Virus Precipitation Solution in the LentiStarter 2 0 Kit provides a simple and highly effective means to concentrate lentiviral particles PEG it is a formulation of polyethylene glycol optimized for the precipitation of lentiviral based particles The PEG it Virus Precipitation Solution is provided as a 5x solution 1 Transfer supernatant containing virus to a sterile vessel and add 1 volume of cold PEG it Virus Precipitation Solution 4 C to every 4 volumes of virus supernatant Example 5ml PEG it with 20ml viral supernatant 2 Refrigerate overnight at least 12 hours Viral supernatants mixed with PEG it Virus Precipitation Solution are stable for up to 4 5 days at 4 C 3 Centrifuge supernatant PEG it mixture at 1500 x g for 30 minutes at 4 C After centrifugation the virus particles may appear as a beige or white pellet at the bottom of the vessel 4 Discard the supernatant into a suitable biohazard waste container Spin down residual PEG it 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 lentiviral particles in pellet 5 Resuspend lentiviral pellets in 1 500 to 1 1000 of original vo
9. apor phase of liquid nitrogen dewar immediately upon arrival IV XPack Purified Exosomes Exosomes have been purified from each stable cell line described above These exosomes are ready to use for addition to target cells for in vivo tracking studies or for other applications Pre packaged purified exosomes containing GFP Cat XPAK120XP 1 or firefly luciferase XPAK130XP 1 from HEK293T cells are available These exosomes have been validated to contain the specified reporter protein by luciferase assay and Western blot and were obtained from cells grown in exosome depleted FBS therefore they are free of contaminating bovine exosomes The XPack purified exosomes are shipped on blue ice 20 C and should be stored at 20 C upon arrival Avoid freeze thawing the Page 22 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 reagents Shelf life of the product is 1 year after receipt if stored in 20 V Frequently Asked Questions Q How long and in what condition should store exosomes after isolation from exosome generating cell line After exosomes are isolated with ExoQuick TC the pellet can be stored at 80 C for 1 year After resuspension in PBS it can be stored at 4 C for 2 weeks or 20 C for 3 months Q How many exosomes should I add to my target cells 250 ug of exosomes as determined by A280 on NanoDrop is sufficient to see efficient delivery
10. ction 2012 PubMed PMID 24009886 PubMed Central PMCID PMC3760650 Vil 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 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 Page 26 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Vill Licensing and Warranty information Limited Use License Use of the XPack system 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 r
11. e by qPCR with SeraMir Kit e Discover novel exoRNA biomarkers with Exo NGS next gen sequencing services lll XPack Stable Cell Lines Stable HEK293T cell lines have been created that constitutively secrete exosomes packaged with GFP Cat XPAK110CL 1 and Firefly Luciferase Cat XPAK120CL 1 These cell lines were generated by transduction of XPack lentiviral particles into each parental cell line Exosomes from XPack stable cell lines have been validated to contain the specified reporter protein by luciferase assay and Western blot A XPack Stable Cell Line Validation Data Exosomes from XPack Stable Cell Lines are Loaded with Reporter Proteins After a stable cell population was generated using puromycin selection exosomes were isolated from HEK293T XPack stable cell line media using ExoQuick TC The exosomes were 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual subjected to A a Western blot to detect GFP presence or B a luminescence assay to detect presence within exosomes Exosomes from untreated HEK293T cells were used as a control A HEK293 XPack GFP Exosomes Exosomes Luminescence in Exosomes Sy 9 c 374 80 om en 70 a 60 EB u E IN Pir gt 5 Y 22 w 10 0 HEK293 Exos HEK293 XPack Luciferase Exos Page 20 ver 1 150316 www systembio com XPack Exosome Targeting System Cat s XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Fig 10 A Exos
12. ee 650 968 2200 outside US Page 13 System Biosciences SBI User Manual Luminescence of Luciferase Packed Exosomes Bo 0 C x 120 2 A My 100 D i c 80 O0 amp 5 So 60 v Ze 40 pw wE g e S 20 0 HEK 293 Exos XPack Luciferase Exos Fig 5 Luminescence assay on exosomes secreted from XPack Luciferase transfected HEK293T cells XPack Proteins are Delivered to Target Cells 250 ug exosomes from XPack GFP transfected HEK293T cells were added to na ve HEK293T target cells in culture in a 6 well plate format 24 hours after exosome addition cells were imaged on a Leica DMI300B fluorescence microscope using a standard GFP filter set Page 14 ver 1 150316 www systembio com XPack Exosome Targeting System Cat s XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 GFP Phase ast 44 GS ison Um Fig 6 Exosomes from HEK293T cells transfected with XPack GFP deliver GFP to target cells as analyzed by fluorescence microscopy 48 hours after exosome addition target cells were lysed and the resulting proteins were analyzed using Western blot probing for GFP XPack GFP Exos GAPDH was used as a cellular marker 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual and a loading control Abcam catalog ab9485 Control cells were incubated with exosomes from non transfected HEK293T cells NT Exos or with exosomes from HEK293T cells transfected with a GFP
13. esold 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 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 888 266 5066 Toll Free 650 968 2200 outside US Page 27 System Biosciences SBI User Manual 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 SBPs liability does not extend to any damages arising from use
14. from cells and are ready for addition to target cells in culture or in vivo studies The XPack stable cell lines have been validated to continuously secrete exosomes containing reporter proteins GFP RFP or luciferase and therefore can be regarding as cellular factories secreting exosomes for downstream applications Finally the purified exosomes from XPack stable cell lines are ready for addition to target cells or in vivo studies Transfection Transduction of Exosome Producer Cells fer XPack Lentiviral Particles Exosome Addition to Target Cells in vivo model Phenotypic Assay Exosome Tracking Study etc Fig 1 Workflow for using XPack lentivectors 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual ll XPack Lentivector System A List of Constructs and Pre Packaged Particles Cat Description Size XPAK100PA 1 XPack Cloning Lentivector 10 ug XPAK110PA 1 XPack GFP Lentivector 10 pg XPAK120PA 1 XPack RFP Lentivector 10 ug XPAK130PA 1 XPack Luciferase 10 ug Lentivector XPAK110VA 1 XPack GFP Pre Packaged 2x 25 ul gt 1x Lentivirus 1046 IFUs XPAK120VA 1 XPack RFP Pre Packaged 2x 25 ul gt 1x Lentivirus 1046 IFUs XPAK130VA 1 XPack Luciferase Pre 2x 25 ul gt 1x Packaged Lentivirus 1046 IFUs NOTE ExoQuick TC and Exo FBS are not provided with the XPack vectors and can be purchased separately The follo
15. lume of pooled virus supernatant using cold sterile Phosphate Buffered Saline PBS or DMEM containing 25mM HEPES buffer at 4 C For example if you performed 2 collections from 2 x 150mm plates 20ml per plate this would be approximately 80ml of media You would resuspend the resulting pellet in 80 160 pl of 1X PBS or DMEM 6 Aliquot in cryogenic vials and store at 80 C until ready for use 7 The resulting pseudoviral particles can be accurately titered using SBI s UltraRapid Global Titering Kit Cat LV961A 1 Page 10 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 http www systembio com lentiviral technology delivery systems ultrarapid overview E Transduction of Pseudoviral Particles into Target Cells For efficient transduction of target cells the negative charges present in the virus envelope protein and the cell surface must be neutralized SBs TransDux reagent provided in the LentiStarter 2 0 Kit is a non toxic proprietory formulation that promotes cell virus contact and subsequent fusion by negating these charges The following protocol can be utilized for delivery of virus to your target cells The following protocol is for infection of target cells in a single well of a 24 well plate if using larger vessels please scale up reagents accordingly Day 1 1 Plate 75 000 cells per well into a single well of a 24 well plate in cell cult
16. omes from HEK293T stable XPack cell lines contain GFP as assayed by Western blot or B Firefly Luciferase as assayed by luminescence assay Promega catalog E1501 Exosomes from XPack Stable Cell Lines Deliver Reporter Proteins to Target Cells Exosomes were isolated from the HEK293T XPack stable cell lines using ExoQuick TC Target HEK293T cells were plated at 100 000 cells well in a 24 well plate 50 ug of HEK293T XPack GFP exosomes were added to target cells and the cells were imaged using the GFP filter set on a Leica DMI300B fluorescence microscope 24 hours after exosome addition GFP Fig 11 Exosomes from HEK293T stable XPack cell lines deliver GFP to target cells as assayed by fluorescence microscopy B XPack Stable Cell Growth Conditions IMPORTANT NOTE Be sure to culture your exosome producer cell lines in media that does not contain standard 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual FBS There are high levels of bovine exosomes present in FBS Instead use SBI s Exo FBS Exosome depleted FBS Media Supplement cat EXO FBS 50A 1 in place of standard FBS media supplements Parental Cell Base Media Supplement s Line HEK293T Dulbecco s 10 Fetal Bovine Modified Serum Antibiotics of Eagle s Medium choice DMEM C Shipping and Storage Conditions The XPack stable cell lines are shipped on dry ice and should be stored in the v
17. on of specific experimental conditions may be needed Page 6 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 C Cloning of XPack Proteins into XPack MCS 1 The XPack MCS has the following sequence with restriction enzyme sites Xhol BamHI Notl EcoRI Nhel and Pstl in bold and underlined CTCGAGtiGGATCCaaGCGGCCGCgaGAATTCtc GCTAGCatCTGCAG 2 To clone fusions in frame note that the XPack tag is 32 nucleotides long at the DNA sequence level making it necessary to add 1 nucleotide to the 5 end of your ORF to generate an in frame fusion Then count the number of nucleotides from the start of the MCS to where first nucleotide in the initial codon of your inserted ORF will fall and add as many nucleotides as needed to make that number a multiple of 3 Add 1 to this number to generate an in frame XPack tag and add that number of nucleotides to your forward PCR primer between the 5 enzyme site and your ORF sequence 3 Make sure that any added nucleotides do not generate a premature stop codon Example Using EcoRI as the 5 restriction site Number of nucleotides from start of MCS to end of EcoRI site 32 Add 1 to make 33 a multiple of 3 Add 1 to keep XPack fusion in frame 1 1 2 Add 2 nucleotides noted as X to forward PCR primer between EcoRI site and ORF priming sequence 5 GAATTC XX ORF site 888 266 5066 Toll Free 650 968 2200 ou
18. overexpression vector GFP Exos NT GFP Exos XPack GFP Exos Exos GFP IB GAPDH Fig 7 Exosomes from HEK293T cells transfected with XPack GFP deliver GFP to target cells as analyzed by Western blot 250 ug exosomes from XPack Luciferase transfected HEK293T cells were added to HEK298T target cells in culture in a 6 well plate format 24 hours after exosome addition cells were lysed and subjected to a luciferase assay Promega catalog E1501 Control cells were either not treated with exosomes Untreated Cells or with exosomes from untransfected HEK293T cells Cells HEK Exos Page 16 ver 1 150316 www systembio com Relative Luminescence Units XPack Exosome Targeting System Cat s XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Luminescence of XPack Luciferase g Target Cells Normalized to Untreated Cells Untreated Cells Cells HEK Exos Cells XPack Luciferase Exos Fig 8 Exosomes from HEK293T cells transfected with XPack Luciferase deliver Luciferase to target cells as analyzed by a luminescence assay Inset live target cells as analyzed by incubation with 150 ng mL D Luciferin XPack Delivered Proteins are Functional Induction of cell cycle arrest in target cells An XPack fusion to the cell cycle inhibitor CDKN1B was transfected into HEK293T cells Exosomes were isolated and a range of 5 ug to 100 ug of XPack CDKN1B exosomes were added 888 266 5066 Toll Free 650 968 2200 ou
19. se contrast GFP Artery GFP Neurons GFP Fig 2 Workflow for generating high titer lentiviral particles Page 8 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 1 Transfection of XPack plasmids into HEK293T or equivalent producer cells a 18 24 hours prior to transfection seed 7 0 8 0 x10 293T cells per 150mm cell culture plate in standard growth media w o antibiotics Cells should be 80 confluent by next day b During transfection day mix 45 ul of pPACKH1 packaging plasmid mix as provided in the LentiStarter 2 0 Kit and 4 5 yg of XPack lentivector in 1 6 ml of serum free DMEM by pipetting c Add 55 ul PureFection into the same tube Vortex for 10 seconds Note If using other transfection reagents e g Lipofectamine 2000 please follow suggested guidelines for 150mm plates d Incubate mixture at room temperature for 15 minutes e Add mixture drop wise to the dish and swirl to disperse evenly throughout the plates f Change the medium 12 hours or next day after transfection g At 48 hours and 72 hours after transfection collect the medium which now contains pseudoviral particles into a 50 ml sterile capped conical centrifuge tube Centrifuge at 3000 x g for 15 minutes at room temperature to pellet cell debris Transfer the viral supernatant into a new tube Caution You are working with infectious pseudoviral particles at this st
20. secretion This xPack peptide sequence has been incorporated into the XPack cloning and expression lentivectors allowing for reporter proteins or any protein of interest to be loaded into exosomes within a producer cell line then delivered to target cells of choice Additionally stable XPack cell lines have been generated that serve as a constant source of exosomes loaded with reporter protein cargo enabling tracking of exosome dynamics in both human cells and in vivo murine models Purified ready to use exosomes from these stable cell lines are also available as part of the comprehensive XPack system B Uses of XPack Technology The XPack technology allows for cell mediated generation of ready to use exosomes packed with any protein of choice These exosomes can then be used to efficiently deliver proteins to target cells to alter or supplement biological pathways or be used to study exosome trafficking in vivo Page 2 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 To generate exosomes containing high levels of any protein of choice the open reading frame sequence for the selected protein is cloned downstream of the XPack peptide in the XPack cloning lentivector which is suitable for transient transfection assays as well generation of lentiviral particles for downstream production of XPack stable cell lines After transfection or transduction exosomes are isolated
21. tside US Page 17 System Biosciences SBI User Manual to HEK293T target cells in culture at 24 hour intervals for a total of 72 hours Exosomes from non transfected cells were used as a control Cell division was quantified using an MTT Assay Millipore catalog CT02 Cell Proliferation in XPack CDKN1B Target Cells 2 T 1 2 c QOD 1 U 2 0 8 v o 5 0 6 ze rA 0 4 o ay 02 5o go 0 8 5 ug Exosomes 50 ug Exosomes 100 ug 5 Exosomes zZ CDKN1B Exosomes M HEK 293 Exosomes Fig 9 Exosomes from HEK293T cells transfected with XPack CDKN1B inhibit cell division as analyzed by a MTT cell growth assay G Shipping and Storage Conditions The XPack lentivectors are shipped on exosomes on blue ice 4 C and should be stored at 20 C upon arrival Avoid freeze thawing the reagents The pre made XPack lentiviral particles are shipped on dry ice and should be immediately stored at 80 C upon arrival Shelf life of either product is 1 year after receipt if stored properly Page 18 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 H Related Products SBI offers a number of exosome research products You can review them here http Awww systembio com exosomes e ExoQuick exosome isolation reagents Exo FBS exosome depleted media supplement e Detect and quantitate exosomes with ExoAB ELISA and EXOCET kits e Purify exosome RNA and profil
22. tside US Page 7 System Biosciences SBI User Manual D XPack Lentiviral Particle Production For researchers looking for sustained long term expression of the XPack construct in their desired cell line the XPack construct can be transfected into HEK293T producer cells and packaged into pseudolentiviral particles for infection of a target cell line The following schematic Fig 2 and the protocol that follows shows the lentiviral production process using the XPack lentiviral vector How to Make High Titer Lentivirus f Your Make Your Lentivector Clone Lentivector cDNA shRNA reporter microRNA etc Construct pPACK _ f Packaging Mix 2 To make lentivirus co transfect your F lentivector plus pPACK packaging mix 4 Packaging Plasmids using PureFection into 293TN packaging cell line 293TN Producer Cells SS Wait 48 72 hours collect supernatant and Cali Gite combine with PEG it virus concentration Medium Containing solution Next day remove supernatant and Viral Particles 5x PEG it resuspend pellet in sterile PBS Solution Fos ets Y E Concentrate Virus Check Titer using Global UltraRapid Titer Kit g using standard cell line ex 293 cells Global Titering Kit Measure Titer by qPCR O Combine the appropriate amount of virus with TransDux and infect your target cells Animal Models Primary Cells Human Embryonic H9 Cells Sy ahaa Sach Mouse Carotid Human Primary Pha
23. ucer cells were visualized on a Leica DMI300B fluorescence microscope using a standard GFP filter set The characteristic localization pattern observed in exosome producer cells is shown in Figure 3 Fluorescence is detected along the cell periphery which indicate budding exosomes and in a brightly fluorescent spot in the cell interior which indicates the MVB ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 Fig 3 Visualization of XPack GFP exosome producing HEK293T cells Exosomal presence of GFP in exosomes secreted from HEK293T XPack GFP producing cells Fig 4 was determined using Western blot using a primary antibody against Turbo GFP antibody Thermo Scientific catalog PA5 22688 An antibody against the cholinesterase transfer protein CETP Abcam catalog ab19012 was used as an exosomal marker XPack GFP Transfected HEK293 Exosomes Non Transfected HEK293 Exosomes CETP Fig 4 Western blot on exosomes secreted from XPack GFP transfected HEK293T cells Exosomal presence of firefly luciferase in exosomes secreted from HEK293T XPack Luciferase producing cells was determined using Promega s Luciferase Assay System Promega catalog E1501 Notably the size of firefly luciferase is 60 KDa indicating that mid to large size proteins can be packaged into exosomes efficiently using the XPack system Fig 5 below 888 266 5066 Toll Fr
24. ure medium Make sure that cells are well dispersed and are not clumped together Include wells for negative non infected cells Note If infecting target cells for the first time or an optimal MOI is not known please titrate virus at varying MOls 1 5 10 and 20 etc to optimize transduction using a positive control virus with a fluorescent marker such as SBI s pre packaged positive transduction control Cat CD511VB 1 Day 2 2 Cells should be between 70 80 confluent Aspirate medium from cells 3 Combine culture medium with TransDux to a 1X final 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual concentration For example add 2 5 ul of TransDux to 500 ul culture medium and then transfer to each well If using other types of transduction reagents e g Polybrene please dilute the reagent to a final working concentration of 2 8 ug ml 4 Add XPack virus at desired MOI to each well and swirl to mix for negative control wells only add media viral transduction reagent Day 3 5 Aspirate off medium and add complete growth medium to cells Day 5 7 Virus should be integrated into the host cell genome by this time and should be expressing the XPack construct for packaging into exosomes F XPack Sample Data XPack Proteins are Packaged into Exosomes XPack GFP was transfected into HEK293T cells as described above GFP localization patterns within exosome prod
25. wing ExoQuick TC products are recommended for exosome concentration prior to addition to target cells Cat Description Size EXOTC10A 1 ExoQuick TC for Tissue 10 ml Culture Media and Urine EXOTC50A 1 ExoQuick TC for Tissue 50 ml Culture Media and Urine Page 4 ver 1 150316 www systembio com XPack Exosome Targeting System Cat 8 XPAK1xxPA VA 1 XPAK1xxCL 1 XPAK1xxXP 1 EXO FBSHI Exosome depleted FBS 50 mi 50A 1 media supplement Heat Inactivated IMPORTANT NOTE Be sure to culture your exosome producer cell lines in media that does not contain standard FBS There are high levels of bovine exosomes present in FBS Instead use SBI s Exo FBS Heat Inactivated Exosome depleted FBS Media Supplement cat EXO FBSHI 50A 1 in place of standard FBS media supplements B XPack Transfection Protocol Transfection of Exosome Producer Cells 1 Seed exosome producer cells in culture dish of choice to reach 70 80 confluency after 24 hours using media compatible with the cells of choice Because standard FBS contains high levels of bovine exosomes be sure to use SBI s Exosome depleted FBS Media Supplement to ensure that exosomes isolated after cell transfection are not contaminated by bovine exosomes Return cells to incubator 24 hours later mix XPack vector with transfection reagent of choice and follow appropriate protocol to achieve transfection of target cells An example transfection reaction
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