EXO-Prep. Product Insert. Kit for exosome isolation from biofluids or cell culture supernatants HBM-EXP-##

Transcription

1 Product Insert HBM-EXP-## Kit for exosome isolation from biofluids or cell culture supernatants This product is for research use only. It is highly recommended to read this users guide in its entirety prior to using this product. Do not use this kit or its components beyond the indicated expiration date.

2 Table of Contents PRODUCT DESCRIPTION 3 PROCEDURE 3 RELATED PRODUCTS 9 TECHNICAL SUPPORT 10 HansaBioMed 2

3 PRODUCT INFORMATION PRODUCT DESCRIPTION About Exosomes Exosomes are small endosome derived lipid nanoparticles ( nm) actively secreted by exocytosis by most living cells. Exosome release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both amount and molecular composition of released exosomes depend on the state of a parent cell. Exosomes have been isolated from diverse cell lines (hematopoietic cells, tumor lines, primary cultures, virus infected cells) as well as from biological fluids in particular blood (e.g. serum and plasma from cancer patients) and other body fluids (bronchoalveolar lavage fluid, pleural effusions, synovial fluid, urine, amniotic fluid, semen, saliva etc). Exosomes have pleiotropic physiological and pathological functions and an emerging role in diverse pathological conditions such as cancer, infectious and neurodegenerative diseases. is a fast and easy method of exosome isolation from biofluids and cell culture supernatants. Method is based on chemical precipitation. Samples are incubated with solution in ice, then exosomes are separated by centrifugation and solubilized in PBS 1X or deionized water. Procedure is easy to perform, no time-consuming (around 1 hour), does not require ultracentrifugation nor expensive laboratory equipment. Isolated exosomes are suitable for a wide range of analyses, such as NTA, protein profiling by using different techniques (western blotting, ELISA, FACS), nucleic acids extraction and profiling of mrna or small RNA markers. Advantages - Protocol easy to perform - No time consuming - No ultracentrifugation required - Isolate exosomes from cell culture supernatants or biofluids - Able to isolate the overall exosome population in a sample - Isolate exosomes from a small volume of sample (plasma/serum 100 ul) - For complex biofluids as plasma, no trombin pretreatment is required - Isolated exosomes are intact and suitable for different downstream analyses Products Volume Catalog Number 5 ml HBM-EXP-B5 for exosome isolation from biofluids (plasma/serum) 10 ml HBM-EXP-B10 20 ml HBM-EXP-B20 for exosome isolation from cell supernatants 25 ml HBM-EXP-C25 50 ml HBM-EXP-C50 for exosome isolation form urine 30 ml HBM-EXP-U25 60 ml HBM-EXP-U50 HansaBioMed 3

4 PROCEDURE 1- PLASMA/SERUM EXOSOME ISOLATION Volume suggested: Fluid Minumum volume required Volume suggested Plasma 100 ul 100 ul Serum 100 ul 250 ul Step A: Sample preparation: - Preclear plasma or serum samples by 3 centrifugation steps - I. 10 at 300 g (save supernatant, discard the pellet) - II. 20 at 1200 g (save supernatant, discard the pellet) - III. 30 at g (save supernatant, discard the pellet) Step B: Exosome isolation: - Add solution to your sample in ratio 1/4 (i.e. 100 ul of plasma + 25 ul of ) - Mix well by pipetting and inverting tube - Incubate on ice for 1 hour - Centrifuge 20 minutes at g (centrifuge can be performed at 4 C or at RT) - Discard the supernatant - Centrifuge for 2 minutes at 1500 g to eliminate entirely the supernatant - Resuspend the pellet in 100 ul* of PBS 1x - Resuspended exosomes can be used for analysis or stored at -20 C. * Volume of resuspension can be defined by the user on the base of downstream analysis. Western blotting:a complex biofluid as plasma presents a high contents of proteins that coprecipitate with exosomes. We recommend to resuspend the pellet in 100 ul of PBS 1X and to quantify the protein contents via BCA or Bradford assay. For WB analysis we suggest to load on the gel more than 30 ug of total protein contents. If serum is used the entire pellet can be resuspended in an appropriate volume of PBS and loaded on the gel (refer to example below). Plasma 110 kda> Serum 110 kda> isolation Alix Alix - Plasma Exosomes isolated from plasma were resuspended in 100 ul of PBS and protein contents quantified by BCA assay. 60 ug of total protein contents were loaded with Laemmli Sample buffer 5X on acrylamide gel. WB performed using anti-alix antibody (Santacruz). Ultracentrifuged exosomes used as control (UC) - Serum Exosomes isolated from serum were resuspended in 24 ul of PBS 1X, and the entire amount was loaded with 6 ul of Laemmli Sample buffer 5X on acrylamide gel. HansaBioMed 4

5 ELISA Ratio to background CD9 expression in exosomes isolated from different volumes of plasma 100 ul 250 ul 500 ul Plasma volumes used for exosome isolation - Plasma and serum: After exosome isolation resuspend pellet in 100 ul of PBS 1X and load the entire amount in a well of an ELISA* plate. Example reports CD9 expression in isolated exosomes from different volumes of plasma. * HBM-precoated immunoplate or HBM quantification kits are suggested for exosome capture and analysis of exosomal protein markers (see Related Products, page 9) Nucleic acids extraction 22,00 - Plasma and serum: CT Values 24,00 26,00 28,00 30,00 32,00 34,00 100ul 500ul 100ul 500ul UC mir16 mir21 mir223 mir451 Exosome pellet can be directly lysed with lysis buffers for nucleic acids extraction. If RNA extraction is performed by using Trizol or similar organic reagents we suggest to resuspend exosomes in PBS, then to add Trizol into the mixture and to proceed with RNA purification HansaBioMed 5

6 2- URINE EXOSOME ISOLATION Volume suggested: 5 ml up to 20 ml of urine Step A: Sample preparation: Preclear urine as indicated - Centrifuge 10 min at 350 g at RT to eliminate cells and protein aggregates. Step B: Exosome isolation: - Add solution to your sample in ratio 1:4 (i.e 5 ml of urine ml of ) - Mix well by pipetting and inverting tube - Incubate on ice for 1 hour - Centrifuge 20 minutes at g (centrifuge can be performed at 4 C or at RT) - Discard the supernatant - Centrifuge for 2 minutes at 1500 g to eliminate entirely the supernatant - Resuspend the pellet in 100 ul* of PBS 1x - Resuspended exosomes can be used for analysis or stored at -20 C. * Volume of resuspension can be defined by the user on the base of downstream analysis. Western blotting For WB the entire pellet can be solubilized in the appropriate volume of PBS 1X and used for analysis (refer to the example below). Urine 110 kda> Alix - Urine Exosomes isolated from 10 ml and 20 ml of urine were resuspended in 24 ul of PBS 1X, and the entire amount was loaded with 6 ul of Laemmli Sample buffer 5X on acrylamide gel. Ultracentrifuged exosomes (30 ug) were used as control. Western blotting performed using anti-alix (Santacruz) HansaBioMed 6

7 ELISA assay Ratio to background CD9 expression on exosomes isolated by from urine ml UC (30 ug) - Urine After exosome isolation resuspend pellet in 100 ul of PBS 1X and load the entire amount in a well of an ELISA* plate. Example reports CD9 expression in isolated exosomes from 10 ml of urine. 30 ug of purified exosomes via ultracentrifuge (UC) were used as control * HBM-precoated immunoplate or HBM Quantification kits are suggested for exosome capture and analysis of exosomal protein markers (see Related Products, page 9) Nucleic acids extraction Exosome pellet can be directly lysed with lysis buffers for nucleic acids extraction. If RNA extraction is performed by using Trizol or similar organic reagents we suggest to resuspend exosomes in PBS, then to add Trizol into the mixture and to proceed with RNA purification 3- CELL CULTURE SUPERNATANT EXOSOME ISOLATION Volume suggested: 1 ml up to 5 ml of cell supernatant Step A: Sample preparation: - Preclear cell supernatant to eliminate cell debris and macrovesicles by 3 centrifugation steps - I. 10 at 300xg (save supernatant, discard the pellet) - II. 20 at 1200xg (save supernatant, discard the pellet) - III. 30 at 10000xg (save supernatant, discard the pellet) Step B: Exosome isolation: - Add solution to your sample in ratio 1:1 (i.e. 1 ml of supernatant + 1 ml of ) - Mix well by pipetting and inverting tube - Incubate on ice for 1 hour HansaBioMed 7

8 - Centrifuge 20 minutes at g (centrifuge can be performed at 4 C or at RT) - Discard the supernatant - Centrifuge for 2 minutes at 1500 g to eliminate entirely the supernatant - Resuspend the pellet in 100 ul* of PBS 1x - Resuspended exosomes can be used for analysis or stored at -20 C. * Volume of resuspension can be defined by the user on the base of downstream analysis. Final exosome yield can be dependent on the cell line used. Different cell lines produce different quantity of exosomes. If exosome yield is poor, increase the volume of medium, mantaining the ratio with 1/1 (2 ml of cell medium + 2 ml of. Western blotting For WB analysis the entire pellet can be solubilized in the appropriate volume of PBS 1X and used for analysis (refer to the example and conditions described for urine). ELISA assay After exosome isolation resuspend pellet in 100 ul of PBS 1X and load the entire amount in a well of an ELISA* plate. *HBM-precoated immunoplate or HBM Quantification kits are suggested for exosome capture and analysis of exosomal protein markers (see Related Products, page 9) Nucleic acids extraction Exosome pellet can be directly lysed with lysis buffers for nucleic acids extraction. If RNA extraction is performed by using Trizol or similar organic reagents we suggest to resuspend exosomes in PBS, then to add Trizol into the mixture and to proceed with RNA purification HansaBioMed 8

9 RELATED PRODUCTS Overview As the first company entirely dedicated to the field of exosomes, we offer a panel of Kits and Reagents for Exosome Research. Ordering information on a variety of reagents and apparatus available from HansaBioMed is provided below. For more information, visit our website at Products Quantity Catalog Number ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Biological fluids ExoTEST Ready To Use Kit for Overall Exosome capture and quantification from Cell culture supernatant ExoTEST Ready To Use Kit for Tumor-derived Exosome enrichment and quantification from Biological fluids Ready to Use Kit Ready to Use Kit Ready to Use Kit HBM-RTK-POF/## HBM-RTK-POC/## HBM-RTK-PTF/## Custommade ExoTEST Ready to Use Kit Ready to Use Kit HBM-RTK-CMK Exosome Total RNA Extraction Kit (Immunobeads, 10 or 20 Reactions) Ready to Use Kit HBM-RNA-BOF-##/# Exosome Total RNA Extraction Kit (Immunoplate) Ready to Use Kit HBM-RNA-POF/# Tumor-derived Exosome Total RNA Extraction Kit (Immunobeads, 10 or 20 Reactions) Ready to Use Kit HBM-RNA-BTF-##/# Tumor-derived Exosome Total RNA Extraction Kit (Immunoplate) Ready to Use Kit HBM-RNA-PTF/# Immunoplates for Overall Exosome capture from Biological fluids 96 wells plate HBM-POF-##/## Immunoplates for Overall Exosome capture from Cell culture supernatant Immunoplates for Tumor-derived Exosome capture and enrichment from Biological fluids Immunoplates for Neural-derived Exosome capture and enrichment from Biological fluids Immunoplates for Glial-derived Exosome capture and enrichment from Biological fluids Immunoplates for Monocytes- and Platelets-derived Exosome capture and enrichment from Plasma samples Immunobeads for Overall Exosome capture from Biological fluids - 0.4, 1 or 4 microns immunobeads size - Simple or Covalent coating Immunobeads for Overall Exosome capture from Cell culture supernatant - 0.4, 1 or 4 microns immunobeads size - Simple or Covalent coating Immunobeads for Tumor-derived Exosome capture and enrichment from Biological fluids - 0.4, 1 or 4 microns immunobeads size - Simple or Covalent coating 96 wells plate HBM-POC-##/## 96 wells plate HBM-PTF-##/## 96 wells plate HBM-PNF-##/## 96 wells plate HBM-PGF-##/## 96 wells plate HBM-PPP-##/## 10 or 20 reactions HBM-BOLF-##/## 10 or 20 reactions HBM-BOLC-##/## 10 or 20 reactions HBM-BTLF-##/## HansaBioMed 9

10 TECHNICAL SUPPORT Get support For the latest services and support information for all locations, go to At the website, you can: Search for user documents, handbooks, certificates of analysis, citations, and other product support documents Access telephone and fax numbers to contact Technical Support and Sales facilities Or contact us at Material Safety Data Sheet (MSDS) Material Safety Data Sheets (MSDSs) are available at Trademarks ExoTEST and HansaBioMed are trademarks of Hansabiomed OÜ..Other brands or product names are trademarks of their respective holders. Research Use All products are sold for research or laboratory use only and are not intended to be administered to humans or used for medical diagnostics. HansaBioMed 10

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12 For the latest product and technical information, visit hansabiomed.eu and exotest.eu HansaBiomed Homepage Online Shop HansaBioMed LLC Akadeemia tee 15, Tallinn, ESTONIA Contact in US Galen Laboratory Supplies P.O. Box 682 Middletown, CT Tel: Fax: Tel: Fax: For support visit or Visit our website and