SBI EXOTC50A-1|外泌體提取試劑盒 現(xiàn)貨|ExoQuick exosome precipitation solution (5 ml), 提取試劑盒

SBI 外泌體提取試劑盒使用方法：

Exosome抽提試劑

Exosome抽提輔助試劑 SBI|外泌體提取試劑盒|代理

問:	ExoQuick或ExoQuick-TC產(chǎn)品，我應(yīng)該選擇哪一款？
答:	ExoQuick試劑適合于富含exosomes的體液樣本，如血清、血漿或者是腹水等； ExoQuick-TC試劑適合于exosomes含量相對(duì)較少的體液，如細(xì)胞培養(yǎng)上清，尿液及腦脊液(CSF)等。
問:	在使用ExoQuick前，生物體液樣本可以是凍存樣本嗎?
答:	冰凍的生物體液樣本只能在冰上融解一次，并使用ExoQuick標(biāo)準(zhǔn)protocol操作。
問:	不同體液樣本中exosome量、以及exosome蛋白和RNAs量是多少？
答:	不同樣本的量各不相同，請(qǐng)參考以下表格：. 體液 樣本體積 顆粒數(shù) 蛋白量 RNA量 血清/血漿 250 ul 6 x10^11 10 mg 250 ng 尿液(一般) 5 ml 5 x10^9 50 ug 25 ng 組織培養(yǎng)上清 10 ml 取決于培養(yǎng)的細(xì)胞種類，以及其分泌exosome的能力。
問:	使用ExoQuick處理樣本后，得到了較多不溶解沉淀，難以進(jìn)行下游實(shí)驗(yàn)，應(yīng)該怎么辦？
答	一般來說，這種情況會(huì)發(fā)生在血清或者血漿樣本中。 在血漿樣本中，包含許多纖維蛋白和纖維蛋白原，這些物質(zhì)也可被ExoQuick沉淀下來。建議在使用Exoquick處理樣本之前，按照操作說明書使用凝血活素D（Thromboplastin D）預(yù)處理樣本。 在血清樣本中，很有可能是ExoQuick處理血清后孵育時(shí)間過長(zhǎng)，或者是離心時(shí)間過長(zhǎng)，請(qǐng)參考以下建議： 生物 體液 預(yù)處理步驟 選擇的ExoQuick產(chǎn)品 樣本體積： ExoQuick體積 孵育時(shí)間 離心速度/時(shí)間 血清 無 ExoQuick 4:1 (v:v) 30 minutes 13,000rpm/2min 血漿 Thromboplastin D處理（請(qǐng)參考說明書） ExoQuick 4:1 (v:v) 30 minutes 13,000rpm/2min  SBI|外泌體提取試劑盒|代理
問:	如何重懸富集得到的exosome沉淀?
答:	這取決于exoosome下游實(shí)驗(yàn)，請(qǐng)參考以下建議： 下游實(shí)驗(yàn) 使用的Buffer Western Blotting RIPA buffer ExoELISA Exosome Binding Buffer 提取RNA RNA提取試劑盒中的裂解Buffer Exosome運(yùn)輸研究 無菌水，PBS，DMEM，或者其他培養(yǎng)基 SBI|外泌體提取試劑盒|代理

ExoQuick-TC?

Exosome Precipitation Solution

Cat# EXOTC10A-1

Cat# EXOTC50A-1

User Manual


Version 7 10/31/2016




Store kit at 4°C on receipt


A limited-use label license covers this product. By use of this product, you accept the terms and conditions outlined in the License and Warranty Statement contained in this user manual.


Contents

Product Description 2

List of Components 2

Storage 2

General Information 2

Protocol: ExoQuick-TC? 3

Next Steps and Related Products 4

Example Data and Applications 5

1. Nanosight 5

2. Urine exosome protein marker analysis 5

Troubleshooting 6

Technical Support 6

Licensing and Warranty Statement 7

Product Description

ExoQuick-TC? is a proprietary polymer that gently precipitates exosomes and microvesicles between 30 and 200 nm in size from tissue culture media, urine, or spinal fluid. First, pre-clear your samples of cells and cellular debris, and then simply add the appropriate amount of ExoQuick-TC to your cleared biofluid, refrigerate, and centrifuge (see the product manual for protocol details). Your exosomes will be in the pellet, ready for resuspension in an appropriate solution.

List of Components

Item Catalog # Volume Reactions

ExoQuick-TC exosome EXOTC10A-1 10 ml Up to 10 reactions
precipitation solution

ExoQuick-TC exosome EXOTC50A-1 50 ml Up to 50 reactions
precipitation solution


Storage

The ExoQuick-TC? kits are shipped at room temperature or on blue ice and should be stored at +4°C upon receipt. Properly stored kits are stable for 1 year from the date received.

General Information

The reaction size is based on using 5 ml of tissue culture media or urine for exosome isolation. For best recovery for both RNA and Protein analysis, we recommend starting with 10 ml sample. Examples of precipitating exosomes from various biofluids can be seen in the Table below.

Bio-fluid Sample volume ExoQuick-TC volume

Tissue Culture media 5 ml/10 ml 1 ml/2 ml

Urine 5 ml/10 ml 1 ml/2 ml

Spinal fluid 5 ml/10 ml 1 ml/2 ml



To isolate exosomes from serum, we recommend using the ExoQuick reagent (cat# EXOQ5A-1 or EXOQ20A-1) which is a distinct formulation from ExoQuick-TC reagent detailed in this manual.

To isolate exosomes from plasma, we recommend using the ExoQuick Plasma Prep and Exosome Precipitation Kit (Cat# EXOQ5TMA-1). Plasma contains fibrin which will precipitate along with ExoQuick causing an insoluble pellet to form. The ExoQuick Plasma Prep and Exosome Precipitation kit contains reagents to help dissolve the fibrin, thus increasing the yield of exosomes precipitated.

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Protocol: ExoQuick-TC?

Note: This protocol is a starting point for most cells in culture. Different cell types secrete different amount of exosomes. You may need to scale this protocol accordingly.

1.Collect biofluid and centrifuge at 3000 × g for 15 minutes to remove cells and cell debris.

2.Transfer supernatant to a sterile vessel and add the appropriate volume of ExoQuick-TC to the bio-fluid. Mix well by inverting or flicking the tube.

3.Refrigerate overnight (at least 12 hours) at +4°C. The tubes should not be rotated or mixed during the incubation period and should remain upright.

4.Centrifuge ExoQuick-TC/biofluid mixture at 1500 × g for 30 minutes. Centrifugation may be performed at either room temperature or +4°C with similar results. After centrifugation, the exosomes may appear as a beige or white pellet at the bottom of the vessel.


5.Aspirate supernatant. Spin down residual ExoQuick-TC solution by centrifugation at 1500 × g for 5 minutes. Remove all traces of fluid by aspiration, taking great care not to disturb the precipitated exosomes in pellet.

6.Resuspend exosome pellet in 100-500 μl using sterile 1X PBS, or specific buffer according to your downstream application. We recommend using the precipitated exosomes immediately rather than freezing them for future use.


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Next Steps and Related Products

Application Related Products Website links

Precipitation of Exosomes from other biological fluids

Exosome Isolation ExoQuick? https://www.systembio.com/microrna-research/exoquick-exosomes/ordering
from Serum


Exosome Isolation ExoQuick? Plasma
prep and Exosome https://www.systembio.com/microrna-research/exoquick-exosomes/ordering
from Plasma
precipitation kit


Protein Characterization of Exosomes

Western blotting Exosome antibodies https://www.systembio.com/microrna-research/exosome-antibody/exoab

Antibody Arrays ExoCheck? Assays https://www.systembio.com/microrna-research/exosome-antibody-arrays

ELISA ExoELISA? Kits https://www.systembio.com/microrna-research/exosome-antibody/elisas

Quantification of Exosomes

Quantification of EXOCET? Assays https://www.systembio.com/microrna-research/exosome-antibody/exocet-assay
exosomes


RNA extraction from Exosomes

RNA extraction and SeraMir? kits https://www.systembio.com/microrna-research/seramir-exosome-rna-
profiling profiling/overview





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Example Data and Applications

1.Nanosight

The NanoSight LM10 instrument is based on a conventional optical microscope and uses a laser light source to illuminate nano-scale particles within a 0.3 ml sample introduced to the viewing unit with a disposable syringe. Enhanced by a near perfect black background, particles appear individually as point-scatterers moving under Brownian motion. The image analysis Nanoparticle Tracking Analysis (NTA) software suite allows users to automatically track and size nanoparticles on an individual basis. Results are displayed as a frequency size distribution graph.

For the NanoSight analysis, 2ml of ExoQuick-TC were combined with 10ml of conditioned media from Human HT1080 lung sarcoma cells or Human embryonic kidney (HEK293) cells. 5ml of normal human urine was combined with 2.5 ml of ExoQuick-TC. All samples were incubated overnight at 4°C for exosome precipitation. The exosomes were resuspended in 1ml of PBS and visualized on the NanoSight LM10 instrument (The HT1080 culture media were diluted 1:40 and the urine sample diluted to 1:50 prior to analysis). HT1080 culture media analysis showed that ExoQuick-TC isolated 133nm (peak)

exosomes with a recovery of 1.74 X109 particles/ml. The HEK293 showed 137nm exosomes with a recovery of 3.33X108 particles/ml. Normal human urine showed 107nm exosomes with a recovery of 4.8X109 particles/ml.


2.Urine exosome protein marker analysis

Ten milliliters of normal human urine was combined with 2ml ExoQuick-TC to precipitate urine exosomes. The exosome pellet was resuspended 175 μl buffer and increasing amounts of the exosome suspension was loaded onto an ELISA-ready plate. The CD9 protein was detected using SBI’s rabbit anti-CD9 primary antibody and SBI’s HRP-conjugated secondary goat anti-rabbit antibody. The size of urine CD9 proteins was determined using Western blot analysis with the same set of antibodies.



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Troubleshooting

I don’t see a pellet after centrifuging my Scale up the volume of tissue culture media to precipitate more
sample exosomes.

The  exosome  pellet  is  difficult  to This can be caused by salt within the ExoQuick-TC and often occurs after
resuspend in buffer freezing exosomal pellets. Try adding slightly more PBS to the pellet.
Alternatively, you can use 0.5x PBS or water, and allow the pellet to sit at
room temperature for 5-10 minutes before resuspending.


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)
2438 Embarcadero Way
Palo Alto, CA 94303

Phone: (650) 968-2200
Toll-Free: (888) 266-5066
Fax (650) 968-2277

E-mail:

General Information: info@systembio.com

Technical Support: tech@systembio.com

Ordering Information: orders@systembio.com


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Licensing and Warranty Statement

Limited Use License

Use of the ExoQuick-TCTM Exosome Precipitation Solution (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:

?The Product shall be used by the purchaser for internal research purposes only. The Product is expressly not designed, intended, or warranted for use in humans or for therapeutic or diagnostic use.

?The Product may not be resold, modified for resale, or used to manufacture commercial products without prior written consent of SBI.

?This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research.

SBI has pending patent applications related to the Product. For information concerning licenses for commercial use, contact SBI.

Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement. Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned. SBI disclaims any and all responsibility 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 refund. This limited warranty shall not extend to anyone other than the original purchaser of the Product. Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product.

SBI’s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price. SBI’s liability does not extend to any damages arising from use or improper use of the Product, or losses associated with the use of additional materials or reagents. This limited warranty is the sole and exclusive warranty. SBI does not provide any other warranties of any kind, expressed or implied, including the merchantability or fitness of the Product for a particular purpose.

SBI is committed to providing our customers with high-quality products. If you should have any questions or concerns about any SBI products, please contact us at (888) 266-5066.


© 2016 System Biosciences (SBI), All Rights Reserved

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System Biosciences (SBI)

2438 Embarcadero Way
Palo Alto, CA 94303

Phone: (650) 968-2200
Toll-Free: (888) 266-5066
Fax: (650) 968-2277

E-mail:

General Information: info@systembio.com

Technical Support: tech@systembio.com

Ordering Information: orders@systembio.com