Science topics: MethodologyLaboratory Techniques and ProceduresCellular Biology TechniquesExosome IsolationScience topicExosome Isolation - Science topicExplore the latest questions and answers in Exosome Isolation, and find Exosome Isolation experts.Questions (147)Publications (391)Questions related to Exosome Isolation12 David Roig-Carlesasked a question related to Exosome IsolationHow can I calculate the iodixanol density when I am running an Optiprep gradient density for exosome isolation?Question5 answersFeb 4, 2016Hello, I am running an Optiprep iodixanol gradient density to isolate my exosomes. With each run I also have a control which I exact 12 fractions and from the protocol it said I need to calculate the density of each fraction so I will know in which might be my exosomes. However, although it might be quite straight forward, I am having difficulties in calculating the density of each fraction. I have been given the coefficient of extinction 320 L g-1 cm-1 and the wavelength 244nm. And with that I dilute each franction to 1:10000 and read the absorbency. However, I do not know how to get to the density from there. Any suggestion?ThanksRelevant answerAishu KrishJul 31, 2021Answer@julien siracusa I have the very same problem. But I have twice higher OD than recommended in all 5,10,20 and 40% fractions. I thought its handling error. So I have repeated again very carefully, no Pipestone error no calculation errors. Yet it s same. If someone knows about this kindly help me to figure out.Thanks in advance View0 Recommendations Julia Bauzá Martinezasked a question related to Exosome IsolationBlack vs cup-shape EVs in TEM?Question4 answersMar 22, 2021Dear all,Does someone know what these black EVs are in my UC preparation from a human cancer cell line (not known to be infected with any virus)?As you can see in the TEM scan, not only we have cup-shaped EVs of various sizes, but also these black/dark vesicles of non-constant diameter. Cells were checked for mycoplasma and did not look infected by anything as they grew normally and looked great under the microscope. Could they just be a vesicle subpopulation? Maybe some protein aggregates?Please let me know your thoughts on the topic.Best regards,Julia JBM_CL1_005.tif4.33 MBRelevant answerKetil Winther PedersenMar 25, 2021Answerhi again, just a few thoughts an early morning :)here is a labeling protocol that I have been usingLabelling protocol- exosome loading undiluted at RT for 15 min- block with 0.5 % BSA 10 min- prim Ab labelling for 30 min- wash 5xPBS 10 min total- R M (1:100) 30 min- wash 5xPBS 10 min total- Prot A Au 10 nm 15 min- wash 5xPBS 10 min total- wash 5xH20 10 min total- embedding in 0.3 % Uranyl acetate in methyl cellulose on ice- TEM analysisNegative Stain1. Glow discharge carbon coated copper grids to improve adsorption by making the surface hydrophilic. T2. Upconcentration of exosomes on the grids can be performed by incubating the grid on a drop of exosomes for 10 min, then remove the grid for a few min and then put it back on the same drop of exosomes again. 3. For double labeling different sizes of protein A gold are used such as 5, 10, 15 or 20 nm.4. In order to obtain the best possible contrast and resolution several different solutions should be tested. (Aqueous Uranyl Acetate: A 1% to 3% solution of uranyl acetate dissolved in water can be used to negatively stain of many samples. The stain has a low pH so this solution is not recommended for particles that are unstable in acid conditions, Neutral Phosphotungstic Acid: A 1% to 3% solution of phosphotungstic acid is made up in water and the pH is adjusted to 7 using sodium hydroxide. This is a useful stain for many samples but is especially good for viruses that dissociate at low pH. The stain produces less contrast than the uranyl acetate. Ammonium Molybdate: Make up a 1% solution of ammonium molybdate in water. This solution has also been used to negatively stain thawed, thin cryosections of fixed cells.5. Remove excess methyl cellulose and uranyl acetate by using a filter paper and place the grid perpendicular to the filter paper. Barely touch the grid to the filter paper and move the grid until no methyl cellulose is removed. It should only be a thin layer of methyl cellulose covering the exosomes on the grid.Stay SafeketilView0 Recommendations Amina Dakeasked a question related to Exosome IsolationSeparation of EVs (Exosomes)Question4 answersDec 7, 2020 Hey, does anyone know any effective way to separate virus from exosomes fraction?my problem with the separation of the EVs in this system is the fact that the virus that we work with particles are more or less the same size as the EVs, ~100nm. Therefore, i will need to apply an additional separation method.we tried separate the EVs from the virus particles, by Optiprep velocity gradient and it didn’t work; we also tried to use iodixanol based gradients .any ideas? thanksRelevant answerNguyen Tien CuongDec 8, 2020Answerhi Amina, I think the size of EV is the same but molecular weight maybe different. In 2019, there is article published in Nature by Zhang et al confirmed that in EV there large exosome, small exosome and exomere. so maybe there are more smaller particles ( 100nm) in EVs than in virus. In my opinion, you can try to extract both EV and virus particles by differential centrifugation, and try to run HPLC to seperate the particles. From there, you can collect fraction and examine which fraction contain virus particles.View6 Recommendations Magdalena Gruntasked a question related to Exosome IsolationNanoSight and Total Exosome Isolation Reagent (Invitrogen)?Question5 answersDec 7, 2018I isolated exosomes from 0.5 ml of human plasma and serum with 2 kits from Invitrogen: Total Exosome Isolation Reagent (from plasma) and Total Exosome Isolation Reagent (from serum). I ve tried to measure the number of partilces and their size by NanoSight, but I didn t succeed. What I could see was some cloudy picture and the screen was grey. From the other hand, I successfuly measured exosomes isolated by ultracentrifugation. I tried to dilute the Invitrogen-isolated samples 1:10, 1:100 and 1:1000, but without success. Does anyone know if there is some special protocol to do it? Or maybe it is not possible at all?Relevant answerJoão Pedro Hübbe PfeiferOct 28, 2020AnswerHi Magdalena,I have been using the Total Exosome Isolation (from cell culture) on my experiments and we have great results on NanoSight300 and TEM, but as you said it is not only exosomes that we isolate, sometimes microvesicles are present as well. I always follow the Invitrogen s protocol recommendations and then I dilute samples to 1:100 in PBS. Regards,João PedroView0 Recommendations Haroon Khanasked a question related to Exosome IsolationCan we conjugate rabies virus glycoprotein (RVG) directly with exosome? Question2 answersOct 21, 2020I have read many articles where researchers get RVG-Exo by transfecting the exosome producing cell through RVG Plasmid. I wanted to ask if anybody knows, can we decorate exosome with RVG directly after isolation? Any reference please...With thanksRelevant answerAllaura ConeOct 27, 2020AnswerI haven t personally, but this paper did. Hope it helps!Article RVG-modified exosomes derived from mesenchymal stem cells re... View8 Recommendations Michal Levyasked a question related to Exosome IsolationBradford assay for exosomes?Question9 answersJul 12, 2016I isolated a large amount (I hope) of exosomes and I need to quantify the membrane proteins with a Bradford assay before doing a WB.  I started off with 70 ml of conditioned medium which I managed to concentrate to about 1 ml before ultracentrifugation. After ultracentrifugation I added 200 ul PBS to my samples. My expectation is that the concentration of exosomes is quite high. So now I need to do a Bradford assay and  I would like to know how to dilute my samples.Relevant answerAbayomi OpadeleOct 27, 2020AnswerDr. Francesc E Borràs I d like to kindly confirm from your response if the appropriate assay type for exosomal protein quantification with nanodrop is 1A/cm = 1 mg/ml.I understand this is a general reference setting for protein solution, but I d like to ask if IgG assay type is also relevant considering the lower detection limit which is about 0.06mg/ml compared to 0.08mg/ml for 1A/cm = 1 mg/ml assay type.Thanks!View0 Recommendations Annele Sainioasked a question related to Exosome IsolationWhich is the best method to extract vesicles from cell cultures? Question7 answersApr 28, 2020I am planning to extract cell secreted vesicles from different type of cell cultures. There are some kit based methods available. However, I am wondering if the classical differential centrifugation methodology is still the best to use. Relevant answerAnnele SainioSep 23, 2020AnswerThank you Alexander Sasha Vlassov and Wasim Ahmed :-)View0 Recommendations Brenda Huangasked a question related to Exosome IsolationWhat do you do with your purified extracellular vesicles?Question3 answersMay 13, 2020Hi all!When you purify extracellular vesicles, such as exosomes, what are your downstream applications/analyses? I m especially interested in understanding what your next steps are if you re doing biomarker discover or diagnostic research. Thanks in advance for your input!Best,BrendaRelevant answerMichael OkparaJun 30, 2020AnswerIn addition, exosomes (from the secretome) could be subjected to mass spectrometry analysis to determine the presence or absence of potential biomarkers in the exosomal fraction. However, it must be stated that the integrity of the exosomes must be assessed either through the use of transmission electron microscopy (TEM) or by confirming the absence of cellular contaminations such as mitochodrion, ER, Golgi bodies etc. View0 Recommendations Serbay Ozkanasked a question related to Exosome IsolationWhat is the quality of the exosomes isolated by using ExoEasy Max kit with respect to purity and quantity?Question6 answersJun 1, 2020What is the quality of the exosomes isolated by using ExoEasy Max kit with respect to purity and quantity?? Is it better than the ExoQuick kit ? I would be very greatful if you share your personal experience...https://www.qiagen.com/ca/products/discovery-and-translational-research/exosomes-ctcs/exosomes/exoeasy-maxi-kit/ https://systembio.com/shop/exoquick-ultra-ev-isolation-kit-serum-plasma/ Relevant answerDimitri AubertJun 4, 2020AnswerHi Serbay,Neither of these kits are optimal for EV isolation, regardless of the source material (biological fluids or cell cultures), UC and SEC are more consistant methods. You can refer to this recent publication, in which the most common methods of extracellular vesicle isolation are compared Article Quality and efficiency assessment of six extracellular vesic... View4 Recommendations Kristina Schiavoneasked a question related to Exosome IsolationHow do you label exosomes with Dil?Question3 answersDec 6, 2016Hi all,I am trying to label purified exosomes (isolated from cell culture media) with Dil (a lipophilic membrane stain) to check for uptake by cells. Has anyone tried this before?I have a few questions regarding the washing step mainly. I ve tried using viva-spin to wash but I lost alot of my exosomes. Would ultracentrifugation work better? And what s the best diluent to use? PBS or Dil C?ThanksKristina Relevant answerNeha RanaApr 29, 2020AnswerHi Kristina , I am wondering about if you used further DiD or DiL dyes for tracking EVs ? Thanks in advance .@KristinaSchiavone View0 Recommendations Tianpeng Zhangasked a question related to Exosome IsolationHow to increase the purity of my exosome to get good quality RNA?Question3 answersFeb 6, 2020 I extracted my exosomes by ultracentrifuge. Because my culture medium is over 150 ml. After 1000g, 10min, 2000g 20 min, I concentrated my culture medium by 100 k centrifugal filter. Then 10000g for 30min, two times 100,000 g for 70min. I extracted my exosomes and other lab exosomes(purity is good, call it the positive sample). The positive sample s yield and purity are fine(OD260/280 is 1.85, 140ng/ul), but my samples are not good(OD260/280 is 1.55, 50ng/ul). The let-7 and U6 snRNA CT values are different in my exosome RNA vs positive control. I think the reason may the purity of my exosomes is not good or RNA. Can someone give me some suggestions to increase the purity of my exosome? Thank you！ Relevant answerAlexandra NordlohneMar 12, 2020AnswerHey Tianpeng,I would also recommend to use a more specific exosome isolation method. Here is a novel exosome isolation technique that delivers higly pure exosomes (without attached antibodies or beads): https://www.iba-lifesciences.com/exosome-isolation.html. You can find application notes there as well to see the quality of the isolated exosomes. Maybe it can help to improve your results.View0 Recommendations Victor Llombartasked a question related to Exosome IsolationCan anyone suggest us a buffer to detach exosomes from magnetic beads?Question18 answersNov 21, 2016We are trying to isolate CD63+ exosomes in order to characterize them by electronic microscopy, Nanosight and ultimately by proteomics/transcriptomics. To isolate them we use the \"Total Exosome Isolation” kit (Invitrogen cat#4478359) obtaining the total exosome population from our cell’s culture media followed by and immunoisolation of the CD63+ exosomes subpopulation using the Exosome – Human CD63 Isolation/Detection kit (Invitrogen cat#10606D). This second kit consists on magnetic beads coated with anti-CD63 antibodies. Our main concern before analysing the particles that we obtain is detaching the exosomes from the beads, especially for Nanosight evaluation. The techsupport team from Invitrogen could not recommend us any buffer but their suggestion was using a solution with a high salt concentration or low pH. Still, they are not sure it will work.Can anyone suggest us a buffer or strategy to detach the exosomes from the beads’ surface?Relevant answerMayra TuicheDec 19, 2019AnswerStefano, yes, I called SBI and they told me that it is possible to purchase only the exoflowbuffer2. You have to call them, it is not possible to order online.My question now is if the exoflowbuffer2 would also dissociate the antibody from the exosome, since in my case, I need the exosomes alone (without bead or antibody) to treat cells.TIA in case anyone knows the answer to that question!View10 Recommendations Salman Fozailasked a question related to Exosome IsolationFor Exosomes isolation, how long should the cells be cultured in serum-free media? Question6 answersOct 9, 2019I have been trying to isolate exosomes from HEK293 cells. and i have read and tried several protocols. but still can t get any exosomes pellet in the final step. How long should i culture cells in serum-free media? Does longer incubation increases yield of exosomes? if someone can provide me with reliable protocol, that would be really helpful. Relevant answerIoannis AzoidisOct 9, 2019AnswerUsually, studies use EV depleted FBS but haven t seen serum free. Also, pellet will probably not be visible in any case especially if you don t culture for long, but it is there (re-suspend where it should be).View10 Recommendations George Shapovalovasked a question related to Exosome IsolationBest lysosome purification kit?Question2 answersJan 20, 2017We need to make a reasonably clean isolation of lysosomes, not necessarily intact, but clear of other organelles/intracellular membranes as much as possible. The most obvious thing to do is to just use a kit, but there are so many. Even a quick search shows most major companies selling one, starting with names like Sigma, Fisher/Thermo and going to many smaller ones..Anybody had any experiences with those kits? Are they noticeably different? Which one is better for purity?Thank you all in advance..Relevant answerQuanzhi LiSep 4, 2019AnswerYou can check this out:https://inventbiotech.com/products/minute%E2%84%A2-lysosome-isolation-kit-for-mammalian-cells-tissues?_pos=1 _sid=9f08116bf _ss=rView0 Recommendations Josina Bunkasked a question related to Exosome IsolationWhich is the better exosome isolation kit for exosome isolation out of plasma? ExoQuick or Total Exosome Isolation Reagent (from plasma)?Question4 answersMay 20, 2019Hey everybody, I would like to isolate exosomes out of plasma. I‘m wondering if somebody has used the ExoQuick (BIC) or the Total exosome isolation reagent (from plasma, ThermoFisher) and can give me a recommendati which one to use.Thanks!Relevant answerMatt JacksonMay 21, 2019AnswerI compare our affinity methods to polymer precip (this is an old technique, developed for viruses first I think), but I don t waste money on these kits. In my opinion, you just need salt, PEG, time, and a centrifuge to make this happen, pennies per sample compared to these kits. See:https://www.nature.com/articles/srep23978View4 Recommendations Ourlad Alzeus Gaddi Tantengcoasked a question related to Exosome IsolationHow to release viable content from extracellular vesicles?Question2 answersApr 15, 2019I would like to know if you have idea how to lyse the membrane of the extracellular vesicle without damaging its contents? I am looking at bacteria transported via EVs and I want to isolate these bacteria inside the EVs and check whether they are still viable. ThanksRelevant answerVetury SitaramamApr 18, 2019AnswerIf your question relates to identification and viability of the (known or unknown)bacteria in EV, why do you have to lyse the EV and inoculate? If you pour them (the EV fraction) into a growth medium containiing a gradient of a non-ionic detergent, at some point the EV should lyse still leaving the bacteria intact. The resultant matt-like growth( if you are lucky) can be harvested appropriately. At least that is a starter!View7 Recommendations Nur Basirah Ghazaliasked a question related to Exosome IsolationRecommendation for Saliva Exosome Isolation KitQuestion3 answersDec 17, 2018Hello, can anybody suggest a kit for saliva exosome isolation that works with sample volume of less than 1.0 ml?Thank youRelevant answerBhairavi VajariaFeb 22, 2019Answerhttp://www.101bio.com/files/manual_P530.pdfView0 Recommendations Mohamed H. Yousefasked a question related to Exosome IsolationFor proteome profiling of exosomes, which is a better sample; plasma or serum?Question5 answersJan 8, 2019If I am to isolate peripheral exosome for analyzing their protein content, which sample type is more advisable, plasma or serum? Relevant answerAlexander Sasha VlassovFeb 13, 2019Answerplasma might be better, but its trickier to isolate clean exosomes from plasma vs serumView0 Recommendations Sami G. Alsabriasked a question related to Exosome IsolationStressing BMSC to collect Exosomes?Question1 answerJan 25, 2019I do have an experiment with BMSC, trying collect exosomes.I am trying to starv the cells with FBS free DMEM media to enhance the release of Exosomes. the cells even in absence of FBS they look unstressd and happy. and they give small amount of Exosomes.I have used the same procedure with other cell type such Keratinocytes and I was able to see how stress they were, and got a good amount of Exosomes. any Idea about thisRelevant answerFilipic BratkoJan 26, 2019AnswerDear Sami G. Alsari,regarding Your problem of Stressing BMSC to collect exosomes, I am adding I think for You needed basic of Extracellular Vesicles (Exosomes), that You can orient yourself in the research about Exosomes.If You need more, please contact me on the adress:Email: Bratko.Filipic@gmail.com--------------------------------------------------------------------------FIGURES are added as file! Nomenclature of EVsThe field of EV (Extracelular Vesicles) research has been hampered not only by lack of standardized nomenclature but also by lack of criteria to distinguish, isolate, and identify the different subtypes of EVs. The term EV encompasses several subtypes of generated cells and expelled vesicles that are enclosed by a membrane bilayer. These include exosomes, microvesicles, and apoptotic bodies (Figure 1). The subtypes can be differentiated by their size, content, and route of intracellular formation. Exosomes are between 25 and 200 nm in diameter and are formed inside multivesicular bodies within the endocytic pathway (Figure 2).33 Here, the endosomal sorting complex required for transport facilitates the inward budding of the plasma membrane, which fills the exosome with cytoplasmic contents and retains membrane proteins specific to the cell of origin.34, 35, 36, 37 At this point, multivesicular bodies may either dock and fuse with the plasma membrane to release exosomes into the intracellular space or deliver their contents to the lysosome for degradation (Figure 2).38, 39 Microvesicles are 100 to 1000 nm in diameter and are formed by the outward budding and pinching of the plasma membrane,40whereas apoptotic bodies ( 1000 nm) are much larger and are formed by apoptotic cell membrane blebbing.41Figure 1Extracellular vesicle (EV) subtypes. Extracellular vesicles are divided into 3 categories based on size, contents, and route of formation. Exosomes, the smallest, originate within multivesicular bodies and carry RNAs, proteins, and lipids. Microvesicles, the next largest, form through outward pinching off of the plasma membrane and also contain RNAs, proteins, and lipids. Apoptotic bodies are formed from dying cells as the plasma membrane blebs to recycle contents. Apoptotic bodies are variable in size and contain cell debris, genomic DNA (gDNA), and proteins.

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