The authors acknowledge the use of instruments at the Shared Instrumentation Core Facility at the Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences. This study was supported by the National Natural Science Foundation of China (NSFC; 82172598), the Natural Science Foundation of Zhejiang Province, China (LZ22H310001), the 551 Health Talent Training Project of Health Commission of Zhejiang Province, China, the Agricultural and Social Development Research Project of Hangzhou Municipal Science and Technology Bureau (2022ZDSJ0474) and Qiantang Interdisciplinary Research Grant.

NOTE: A schematic of the method is shown in Figure 1.
1. EM preparation and isolation
<ol>
	<li>Cell internalization of MNPs
	<ol>
		<li>Cell culture
		<ol>
			<li>Suspend 1 &#215; 106 rat bone marrow mesenchymal stem cells (BMSC), 293T cells, or Mouse ovarian epithelial cancer cells (ID8) in DMEM complete medium with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin solution (P/S) in 2 mL per six-well plate (see Table of Materials).</li>
			<li>Culture the cells overnight at 37 &#176;C and 5% CO2. 
			NOTE: The number of cells after adherence was about 1 &#215; 106.</li>
		</ol>
		</li>
		<li>Endocytosis of MNPs
		<ol>
			<li>Reduce the medium volume of each six-well plate to 1 mL. Co-culture the cells with 10 nm polylysine-modified iron oxide nanoparticles (IONPs) (see Table of Materials) at a concentration of 100 &#181;g/1 &#215; 106 cells and incubate at 37 &#176;C in an atmosphere containing 5% CO2 for 12 h to allow the cells to endocytose IONPs fully.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolation and homogenization of endosomes
	<ol>
		<li>Cell hypotonic treatment
		<ol>
			<li>Digest the cells that have fully internalized IONPs with 0.5 mL/well trypsin for 3 min, and terminate the digestion by adding 1 mL/well complete medium.</li>
			<li>Centrifuge at 1000 x g for 5 min, and discard the supernatant. Resuspend the cells in phosphate buffer saline (PBS) and centrifuge, repeatedly twice to wash out the residual medium and unabsorbed IONPs.</li>
			<li>Finally, resuspend the pellet in 8 mL in pre-prepared hypotonic solution (71.4 mM potassium chloride, 1.3 mM sodium citrate, pH 7.2-7.4) for 15 min (see Table of Materials).</li>
		</ol>
		</li>
		<li>Release of organelles by low-speed homogenization
		<ol>
			<li>Transfer the cell suspension in hypotonic solution to a glass test tube and release the organelles using a glass homogenizer (see Table of Materials) by 20 shocks at 1000 rpm.</li>
		</ol>
		</li>
		<li>Magnetic separation to obtain endosomes
		<ol>
			<li>Transfer 1 mL of the homogenized cell solution to a 1.5 mL microcentrifuge tube and place in a magnetic separator for 1 h to fully separate IONP-loaded endosomes from other organelles (such as nucleus and mitochondria) and cell debris.</li>
			<li>Collect the brown pellets on the contact surface of the microcentrifuge tubes next to the magnetic frame (see Table of Materials), i.e., the IONP-loaded endosomes. Discard the liquid in the microcentrifuge tubes and add 3 mL of PBS to resuspend the endosomes.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>High-speed homogenization
	<ol>
		<li>Transfer the solution containing the endosomes to a 15 mL centrifuge tube with a liquid volume between 3-10 mL.</li>
		<li>Extend the 10 mm probe of the high-speed homogenizer (see Table of Materials) to the bottom of the centrifuge tube without touching the bottom. Adjust the Speed button under the screen, set the speed to 140 x g, and adjust the Time button to set the time of 5 min, then press the OK button.</li>
		<li>Press the Start button to carry out the homogenization after placing an ice box under the sample.</li>
	</ol>
	</li>
	<li>Magnetic sorting for EMs
	<ol>
		<li>Transfer the solution containing nanovesicles obtained from high-speed homogenization into the 1.5 mL microcentrifuge tube and put it in a magnetic separator for 1 h.</li>
		<li>Collect the liquid that contains the required EMs. Be careful not to touch the surface of the tubes next to the magnetic frame, which adsorbed free IONPs and IONP-loaded EMs.</li>
	</ol>
	</li>
</ol>
2. EM characterization (Figure 2 and Figure 3)
<ol>
	<li>Dynamic light scattering (DLS) analysis
	<ol>
		<li>Inject 1 mL of EMs sample (20 &#181;g/mL) along the wall into the cuvette and place it in the instrument sample slot of the DLS instrument (see Table of Materials).</li>
		<li>Open the DLS software, select Particle Size Test, and start testing.</li>
		<li>Measure the protein concentration using a BCA protein assay kit (see Table of Materials).</li>
	</ol>
	</li>
	<li>Nanoparticle tracking analysis (NTA)
	<ol>
		<li>Dilute EMs with PBS to a solution at 1 &#215; 107-1 &#215; 108 particles/mL and inject them into the chamber of the NTA instrument (see Table of Materials) using a 1 mL syringe at a flow rate of 500-1000 &#181;L/min.</li>
		<li>Open the 488 laser channels and ensure the number of EMs in the software interface is within 100-300 and in Brownian motion.</li>
		<li>According to the manufacturer&#39;s protocol, select the appropriate SOP (EV-488) to ensure the instrument&#39;s sensitivity is suitable for EM detection.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Fix EMs (1 mg/mL) with an equal volume of 5% glutaraldehyde for 30 min, and quantify the concentration of EMs as 1 mg/mL by BCA protein assay kit.</li>
		<li>Place a 10 &#181;L drop of EMs on the carbon side of the copper mesh, and remove excess liquid from the side with filter paper after 10 min. Add 10 &#181;L of PBS and blot immediately with filter paper. Repeat twice to remove excess glutaraldehyde.</li>
		<li>Drop 5 &#181;L of uranyl acetate contrast agent and negatively stain for 1 min, then remove the excess contrast agent. Wash three times with deionized water and dry at room temperature (RT).</li>
		<li>Record image by a TEM at an acceleration voltage of 100 kV.</li>
	</ol>
	</li>
	<li>Western blotting
	<ol>
		<li>Add 100 &#181;L of cell lysis buffer and 5 &#181;L of a 50x protease inhibitors cocktail to the samples (see Table of Materials). Mix gently with a pipette gun and put on ice for 30 min.</li>
		<li>Centrifuge the solution at 1000 x g for 10 min at 4 &#176;C, quantify the protein concentration of the supernatant with a BCA protein assay kit, and collect it.</li>
		<li>Mix the 100 &#181;L supernatant with 25 &#181;L of 5x protein loading buffer and heat at 95 &#176;C for 10 min.</li>
		<li>Prepare gels according to the instructions of the preformed gels. Load 15 &#181;g of protein per well and run by gel electrophoresis at 80 V. Wait for the sample to run to the separation gel, change to 100 V, and then transfer the protein to a nitrocellulose membrane at 100 V, 60 min under ice bath.</li>
		<li>Detect the non-EV-specific markers (Calnexin) and EV biomarkers (Annexin and CD63) by western blotting (see Table of Materials). 
		&#8203;NOTE: The antibodies are diluted as per the manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
</ol>
3. In vitro EM function detection
<ol>
	<li>EMs labeling
	<ol>
		<li>Mix 1 &#181;L of PKH26 with 250 &#181;L of diluent C (1:250) to make 2x staining solution (4 &#215; 10-6 M) (see Table of Materials).</li>
		<li>Mix 250 &#181;L of EMs (1 mg/mL) with 250 &#181;L of 2x staining solution and quickly blow with a pipetting gun.</li>
		<li>Incubate at RT for 2-5 min while gently inverting the centrifuge tube.</li>
		<li>Add an equal volume of serum or 1% bovine serum protein and incubate for 1 min to terminate the digestion.</li>
		<li>Remove excess PKH26 by ultrafiltration with 1000 KD ultrafiltration tubes at 3000 x g for 30 min, and repeat three times by adding PBS. Resuspend the remaining liquid to 500 &#181;L with PBS and filter through a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Cells uptake assay
	<ol>
		<li>Cell inoculation: Seed 1 &#215; 106 cells in 35 mm confocal dishes with 1 mL of DMEM containing 10% FBS and 5% P/S, and incubate at 37 &#176;C and 5% CO2 overnight.</li>
		<li>Filter the EMs with 0.22 &#181;m sterile syringe filters to remove potential contamination.</li>
		<li>Treat the cells with PKH26-labeled EMs (109 particles/mL) for 8 h.</li>
		<li>Wash three times with PBS, add DAPI (10 ng/mL), and incubate for 10 min at RT.</li>
		<li>Acquire the fluorescence images in confocal laser scanning fluorescence microscopy using the 405 nm and 561 nm laser channels (see Table of Materials).</li>
	</ol>
	</li>
</ol>

Optimized Magnetic High-Yield Production of Endosome-Derived Nanovesicles

<ol>
	<li>Kalluri, R., LeBleu, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology,+function,+and+biomedical+applications+of+exosomes.">The biology, function, and biomedical applications of exosomes.</a> Science. 367 (6478), eaau6977 (2020).</li><li>Hyenne, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAL-1+controls+multivesicular+body+biogenesis+and+exosome+secretion.">RAL-1 controls multivesicular body biogenesis and exosome secretion.</a> J Cell Biol. 211 (1), 27-37 (2015).</li><li>Farooqi, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+biogenesis,+bioactivities+and+functions+as+new+delivery+systems+of+natural+compounds.">Exosome biogenesis, bioactivities and functions as new delivery systems of natural compounds.</a> Biotechnol Adv. 36 (1), 328-334 (2018).</li><li>Gatti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvesicles+derived+from+human+adult+mesenchymal+stem+cells+protect+against+ischaemia-reperfusion-induced+acute+and+chronic+kidney+injury.">Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.</a> Nephrol Dial Transplant. 26 (5), 1474-1483 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome:+A+review+of+its+classification,+isolation+techniques,+storage,+diagnostic+and+targeted+therapy+applications.">Exosome: A review of its classification, isolation techniques, storage, diagnostic and targeted therapy applications.</a> Int J Nanomedicine. 15, 6917-6934 (2020).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+opportunity,+and+perspective+on+exosome+isolation+-+efforts+for+efficient+exosome-based+theranostics.">Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.</a> Theranostics. 10 (8), 3684-3707 (2020).</li><li>Castilletti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+induction+of+IFN-alpha+and+-gamma+by+SARS-CoV+also+in+the+absence+of+virus+replication.">Coordinate induction of IFN-alpha and -gamma by SARS-CoV also in the absence of virus replication.</a> Virology. 341 (1), 163-169 (2005).</li><li>Guo, P., Huang, J., Moses, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+nanomedicines+in+an+evolving+oncology+landscape.">Cancer nanomedicines in an evolving oncology landscape.</a> Trends Pharmacol Sci. 41 (10), 730-742 (2020).</li><li>Jo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+generation+of+cell-derived+nanovesicles.">Large-scale generation of cell-derived nanovesicles.</a> Nanoscale. 6 (20), 12056-12064 (2014).</li><li>Yoon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+nanovesicles+with+sliced+cellular+membrane+fragments+for+exogenous+material+delivery.">Generation of nanovesicles with sliced cellular membrane fragments for exogenous material delivery.</a> Biomaterials. 59, 12-20 (2015).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+mimetics+derived+from+bone+marrow+mesenchymal+stem+cells+ablate+neuroblastoma+tumor+in+vitro+and+in+vivo.">Exosome mimetics derived from bone marrow mesenchymal stem cells ablate neuroblastoma tumor in vitro and in vivo.</a> Biomater Adv. 142, 213161 (2022).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+mimetics+derived+from+bone+marrow+mesenchymal+stem+cells+deliver+doxorubicin+to+osteosarcoma+in+vitro+and+in+vivo.">Exosome mimetics derived from bone marrow mesenchymal stem cells deliver doxorubicin to osteosarcoma in vitro and in vivo.</a> Drug Deliv. 29 (1), 3291-3303 (2022).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+proteomic+analysis+of+exosome+mimetic+vesicles+and+exosomes+derived+from+human+umbilical+cord+mesenchymal+stem+cells.">Comprehensive proteomic analysis of exosome mimetic vesicles and exosomes derived from human umbilical cord mesenchymal stem cells.</a> Stem Cell Res Ther. 13 (1), 312 (2022).</li><li>Li, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+exosomes+for+translational+nanomedicine.">Artificial exosomes for translational nanomedicine.</a> J Nanobiotechnology. 19 (1), 242 (2021).</li><li>Yang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics+of+310+SARS-CoV-2+Omicron+variant+patients+and+comparison+with+Delta+and+Beta+variant+patients+in+China.">Clinical characteristics of 310 SARS-CoV-2 Omicron variant patients and comparison with Delta and Beta variant patients in China.</a> Virol Sin. 37 (5), 12 (2022).</li><li>Shapira, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+platform+for+attenuating+immune+hyperactivity+using+EXO-CD24+in+COVID-19+and+beyond.">A novel platform for attenuating immune hyperactivity using EXO-CD24 in COVID-19 and beyond.</a> EMBO Mol Med. 14 (9), 15997 (2022).</li><li>Jang, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+exosome-mimetic+nanovesicles+for+targeted+delivery+of+chemotherapeutics+to+malignant+tumors.">Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors.</a> ACS Nano. 7 (9), 7698-7710 (2013).</li><li>Le, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+and+mild+isolation+of+intact+lysosomes+using+magnetic-plasmonic+hybrid+nanoparticles.">Quick and mild isolation of intact lysosomes using magnetic-plasmonic hybrid nanoparticles.</a> ACS Nano. 16 (1), 885-896 (2022).</li><li>Jeong, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanovesicles+engineered+from+ES+cells+for+enhanced+cell+proliferation.">Nanovesicles engineered from ES cells for enhanced cell proliferation.</a> Biomaterials. 35 (34), 9302-9310 (2014).</li><li>Kooijmans, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Display+of+GPI-anchored+anti-EGFR+nanobodies+on+extracellular+vesicles+promotes+tumour+cell+targeting.">Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting.</a> J Extracell Vesicles. 5, 31053 (2016).</li></ol>

D.W. and P.G. are co-inventors of a patent application filed by the Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences. The other author declares no conflicts of interest.

As a surrogate of cell-free therapy and a nanoscale drug delivery system, EVs have yet to meet their clinical expectations, and a main obstacle is the lack of scalable and reproducible production and purification methods6. Therefore, various types of EMs have been developed as EV analogs with similar biological complexity14. To date, the most commonly used EM example is cell plasma membrane-derived nanovesicles. The preparation of such nanovesicles is relatively easy and straightforward by directly extruding whole parent cells17. However, cell plasma membrane-derived nanovesicles cannot recapitulate native EVs owing to two drawbacks: First, the biological origin of these nanovesicles is from cell plasma membrane, which contains different lipid and protein compositions in comparison with native EVs; Second, the contaminations, including proteins, nucleic acids and lipids from non-EV organelles and cell debris, causing inevitable EM heterogeneity. In this method, we incubated cells with IONPs and collected IONP-loaded endosomes through magnetic separation, then generated monodisperse nanovesicles via high-speed homogenization, eventually obtaining IONP-free EMs by a second round of magnetic separation. Moreover, we optimized the EM preparation conditions by exploring the impact of different parameters, such as homogenization speed and time, on EM size and yield. This protocol takes advantage of an interesting biological phenomenon: MNPs (e.g., 10 nm IONPs) can be efficiently internalized by almost all cells via endocytosis and exclusively accumulated in endosomes, not other organelles. This unique biological process enabled the isolation of MNP-loaded endosomes without non-EV contaminations via magnetic separation. In return, nanovesicles prepared by these purified cell endosomes could more faithfully recapitulate the biological complexity of native EVs.
Moreover, the golden standard of the EV isolation method is ultracentrifugation, which costs massive cell culture media and a long processing time of over 5 h and only produces 1 &#215; 107-1 &#215; 108 particles from 1 x 106 cells6. Traditional whole parental cell extrusion methods include multiple steps involving ultra-high pressure membrane extrusion and ultracentrifugation, which have a relatively higher yield but require expensive equipment17. However, the EM production yield, efficiency, cost, and manpower are substantially improved by utilizing our protocol. The high-speed homogenizer is a common and low-cost equipment for a short processing time of 5 min in this protocol, and this method can produce up to 100 times more EMs from 1 &#215; 106 cells in comparison with native EVs. However, this method has some limitations, such as loss of endosomal proteins during high-speed homogenization, and it has been shown that MNPs endocytosed by cells can be transferred to lysosomes, which would be a potential contamination18.
In perspective, EMs can be engineered to exert important biological functions as their native counterparts, which may serve as a novel and promising type of biological therapeutics. Bioactive substances (e.g., proteins and nucleic acids) can be integrated into EMs via selecting specific parental cells (e.g., stem cells19) or genetic modification (e.g., fusion proteins20). For example, cell-derived nanovesicles by extruding living embryonic stem cells have a positive effect on the recovery or wound-healing process19. Genetic modification is an alternative approach to generate&#160;EMs with specific biological functions. For example, when cells were transfected with vectors of anti-epidermal growth factor receptor (EGFR) nanobodies fused to glycosylphosphatidylinositol (GPI) anchor signal peptides, the EGFR nanobodies can be anchored on the surface of EVs for targeting EGFR-expressing tumor cells20. EMs have performed well as a surrogate of cell-free therapy and a nanoscale drug delivery system in multiple preclinical studies14, but there is a lack of a comprehensive mechanistic understanding of EM or EV-based therapy with concerns about the heterogeneity and reproducibility of EVs and EMs in clinic investigations. Taken together, the biological functions of EMs and their clinical implications warrant further investigations.

Extracellular vesicles (EVs) are small vesicles secreted by almost all cells with a size range of 30-150 nm, containing abundant bioactive substances. Depending on the cell of origin, EVs show high heterogeneity, possessing multiple components specific to parent cells1. EVs are released into body fluids and transported to distant sites where they are taken up by target cells for action2, which can be utilized to deliver a wide range of bioactive molecules and drugs for tissue repairing, tumor diagnosis and treatment, and immune modulation3,4. However, other biological nanoparticles (e.g., lipoproteins) and nanovesicles (e.g., EVs derived from non-endosomal pathways) with similar biophysical properties in body fluids inevitably affect EV isolation and purification. To date, ultracentrifugation remains the gold standard for EV isolation, and other isolation methods, including sucrose density gradient centrifugation, ultrafiltration, polyethylene glycol precipitation, chromatography, and immunomagnetic bead isolation, have been developed5. The current bottleneck limiting clinical translation and commercialization of EV therapeutics is the severe lack of isolation techniques that allow for highly scalable and reproducible isolation of EVs6,7,8. Traditional EV isolation techniques (e.g., ultracentrifugation and size exclusion chromatography) suffer from low yield (1 x 107-1 x 108/1 x 106 cells), long production cycle (24-48 h), poor reproducibility of product quality, and require expensive and energy-intensive production equipment that cannot meet the current clinical demand for EVs6.
Exosome mimics (EMs), synthetic surrogates of native EVs, have attracted important attention due to their highly similar structure, function, and scalability in production. The main source of EMs is from the direct extrusion of whole parental cells with continuous sectioning9,10, demonstrating potent biological functions as native EVs11,12. For instance, EMs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) exert similar wound-healing effects as native EVs and are richer in protein composition13. Though EMs derived from whole cells have the biological complexity of EVs, their main drawback is the heterogeneity of products because they are inevitably contaminated by various cellular organelles and cell debris. Protein localization analysis further revealed that EMs derived from whole-cell extrusion contain many non-EVs-specific proteins from mitochondria and the endoplasmic reticulum13. Moreover, most methods for manufacturing EMs still require ultracentrifugation, a highly time and energy-consuming process14. Considering the fact that exosomes are exclusively derived from cellular endosomes, we hypothesized that bioengineered endosome-derived nanovesicles may better recapitulate the biological homology between exosomes and EMs in comparison with the well-established cell membrane-derived EMs produced by whole cell extrusion method14. Nevertheless, the manufacture of endosome-derived nanovesicles is difficult due to the lack of viable approaches.
Clinical studies have been carried out by utilizing EVs as a surrogate of cell-free therapy and a nanoscale drug delivery system for the treatment of various diseases. For instance, EVs derived from bone marrow mesenchymal stem cells have been used to treat severe pneumonia caused by COVID-19 and have achieved promising results. Recently, genetically engineered EVs carrying CD24 proteins have also demonstrated potent therapeutic benefits for treating COVID-19 patients15,16. However, the clinical requirement of EV therapy still cannot be met with traditional isolation methods because of the low yield and cost. This study reports the large-scale production of endosome-derived nanovesicles via a magnetic separation-assisted high-speed homogenization approach. It takes advantage of the endocytosis pathway of MNPs to isolate MNP-loaded endosomes via magnetic separation, followed by high-speed homogenization to formulate endosomes into monodisperse nanovesicles. Since the types of endosomes collected by this protocol are diverse, further in-depth research is still required to establish good manufacturing practices (GMP) in the industry. This novel EM preparation approach is more time efficient (5 min of high-speed homogenization) to obtain nanovesicles homologous to native EVs. It produces exponentially more vesicles from the same amounts of cells than ultracentrifugation, which can be generally applied to various cell types.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Annexin antibody</td>
			<td>ABclonal</td>
			<td>A11235</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Beyotime</td>
			<td>P0012</td>
			<td>Protein concentration assay</td>
		</tr>
		<tr>
			<td>Calnexin</td>
			<td>GeneTex</td>
			<td>HL1598</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>ABclonal</td>
			<td>A19023</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>Cell lysis buffer for Western and IP</td>
			<td>Beyotime</td>
			<td>P0013</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>Allegra X-30R</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo</td>
			<td></td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Confocal laser scanning fluorescence microscopy</td>
			<td>NIKON</td>
			<td>A1 HD25</td>
			<td>Photo the fluorescence picture</td>
		</tr>
		<tr>
			<td>DMEM basic (1x)</td>
			<td>GIBCO</td>
			<td>C11995500BT</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Dynamic light scattering (DLS)</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS ZEN3600</td>
			<td>Diameter analysis</td>
		</tr>
		<tr>
			<td>Electric glass homogenizer</td>
			<td>SCIENTZ(Ningbo, China)</td>
			<td>DY89-II</td>
			<td>Low-speed homogenization</td>
		</tr>
		<tr>
			<td>Exosome-depleted FBS</td>
			<td>system Bioscience</td>
			<td>EXO-FBS-50A-1</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>High-speed homogenizer</td>
			<td>SCIENTZ(Ningbo, China)</td>
			<td>XHF-DY</td>
			<td>High-speed homogenization</td>
		</tr>
		<tr>
			<td>Magnetic grate</td>
			<td>Tuohe Electromechanical Technology (Shanghai, China)</td>
			<td>NA</td>
			<td>Magnetic separation</td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich</td>
			<td>PKH26GL-1KT</td>
			<td>The kit contains PKH26 cell linker in ethanol and Diluent C</td>
		</tr>
		<tr>
			<td>Polylysine-modified iron oxide nanoparticles (IONPs)</td>
			<td>Zhongke Leiming Technology (Beijing, China)</td>
			<td>Mag1100-10</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Aladdin</td>
			<td>7447-40-7</td>
			<td>Cell hypotonic treatment</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Beyotime</td>
			<td>P1030</td>
			<td>Proteinase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Aladdin</td>
			<td>7447-40-7</td>
			<td>Cell hypotonic treatment</td>
		</tr>
		<tr>
			<td>Transmission electron microscopy (TEM)</td>
			<td>JEOL</td>
			<td>JEM-2100plus</td>
			<td>Morphology image</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>Optima XPN-100</td>
			<td>Exosome centrifuge</td>
		</tr>
		<tr>
			<td>ZetaView nanoparticle&#160; tracking analyzers</td>
			<td>Particle Metrix</td>
			<td>PMX120</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
	</tbody>
</table>

a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived vesicles

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, their clinical translation has been limited by the lack of scale-up manufacturing approaches. Therefore, this protocol provides a magnetic separation-assisted high-speed homogenization method for the large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) derived from the endosomes, which have about 100-time higher yield than conventional ultracentrifugation method. In this method, magnetic nanoparticles (MNPs) were internalized by parental cells via endocytosis and were subsequently accumulated within their endosomes. Then, MNPs-loaded endosomes were collected and purified by hypotonic treatment and magnetic separation. A high-speed homogenizer was utilized to break MNP-loaded endosomes into monodisperse nanovesicles. The resulting endosome-derived vesicles feature the same biological origin and structure, characterized by nanoparticle tracking analysis, transmission electron microscope, and western blotting. Their morphology and protein composition are similar to native EVs, indicating that EMs may potentially serve as a low-cost and high-yield surrogate of native EVs for clinical translations.

The workflow of EM preparation by magnetic separation-assisted high-speed homogenization is shown in Figure 1. Cells internalize 10 nm polylysine-modified IONPs, which are specifically accumulated in endosomes via endocytosis (Figure 3A). After being treated with hypotonic buffer and homogenized, the IONP-loaded endosomes are released from the cells and subsequently collected by magnetic separation. The isolated endosomes are further reconstituted into monodisperse nanovesicles, also known as EMs, by high-speed homogenization. We explored multiple key parameters (e.g., homogenization speed and time) to identify optimized EM preparation conditions&#160;(Figure 2). Finally, a homogenization speed of 140 x g for 5 min was chosen as the optimized condition by considering the particle size and yield of produced EMs. Free IONPs and IONP-loaded EMs are eventually removed from the final product solution by a second round of magnetic separation to obtain IONP-free EMs. The method produces highly uniform and monodispersed nanovesicles from parental cell endosomes, sharing the same biological origin as native EVs.
To compare EVs obtained by ultracentrifugation with EMs generated by this method, BMSC and 293T were prepared for EVs and EMs. The diameter and morphology of EMs were analyzed by NTA and TEM. The morphology of BMSC-EMs has the feature of a typical bowl-shaped vesicle-like structure and is delimited by a lipid bilayer (Figure 3A). As analyzed by NTA, both BMSC-EMs and 293T-EMs have a similar hydrodynamic diameter to native EVs (BMSC-EVs and 293T-EVs) (Figure 3B). The BMSC-EMs yields of high-speed homogenization were 8.16 &#215; 108-1.42 &#215; 109/1 &#215; 106cells, and the yield of native EVs prepared by ultracentrifugation was only 7.2 &#215; 107-1.12 &#215; 108/1 &#215; 106cells. Similarly, the 293T-EMs yields of high-speed homogenization were 3.71 &#215; 108-7.58 &#215; 108/1 &#215; 106cells, which reaches up to approximately 100-fold higher than those of native 293T-EVs prepared by the conventional ultracentrifuge method (&#8776;5.5 &#215; 106/1 &#215; 106 cells) (Figure 4A).
Moreover, western blotting results showed that BMSC-EMs contain the same protein biomarkers as EVs (CD63 and Annexin). Both EMs and EVs are negative for Calnexin expression, suggesting that EMs produced by this method had almost no plasma membrane contamination (Figure 3C).&#160;There is no significant difference in protein concentration between EM and EV, BMSC-EMs and BMSC-EVs exhibited similar total protein concentrations, 11.15 &#181;g/1 &#215; 109 particles and 14.71 &#181;g/1 &#215; 109 particles via the BCA protein assay kit. Moreover, 293T-EMs and 293T-EVs exhibited total protein concentrations of approximately 31.8 &#181;g/1 &#215; 109 particles and 9.95 &#181;g/1 &#215; 109 particles, respectively (Figure 4B). These results indicate that EMs have a similar protein composition as native EVs. To detect whether EMs can be endocytosed, PKH26-labeled EMs and EVs were co-incubated with BMSC and ID8 at a concentration of 1 &#215; 109 particles/mL for 8 h, and the cells were observed under confocal fluorescence microscopy to confirm that EMs could be readily taken up by the cells for action as well as EVs&#160;(Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66021/66021fig01.jpg" /> 
Figure 1: Schematic diagram of the magnetic-assisted high-speed homogenization method. Step 1: Cells internalize IONPs into endosomes through endocytosis. Step 2: Collect organelles, including IONP-loaded endosomes, after hypotonic treatment and homogenization. Step 3: Purify IONP-loaded endosomes by magnetic separation. Step 4: The endosomes are homogenized and reconstituted into monodisperse nanovesicles. Step 5: Remove free IONPs and IONP-loaded EMs by magnetic separation to collect IONP-free EMs. <a href="https://www.jove.com/files/ftp_upload/66021/66021fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66021/66021fig02v2.jpg" /> 
Figure 2: The optimization of EM preparation conditions. (A) The diameter and PDI of BMSC-EMs in response to homogenization speed changes when time is set at 5 min were analyzed by DLS. (B) The diameter and PDI of BMSC-EMs in response to homogenization time changes when the homogenization speed is set at 140 x g were analyzed by DLS. (C) The diameter and PDI of BMSC-EMs at different storage time points were analyzed by DLS. (D) The diameter and concentration of BMSC-EMs in response to homogenization speed changes when time is set at 5 min were analyzed by NTA. (E) The diameter and concentration of BMSC-EMs in response to the homogenization time change when the homogenization speed is set at 140 x g were analyzed by NTA. (F) The diameter and yield of BMSC-EMs in response to BMSC cells co-incubation with IONPs of different concentrations were analyzed by NTA. ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/66021/66021fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66021/66021fig03.jpg" /> 
Figure 3: Characterization&#160;of EMs. (A) TEM characterization of the morphology of parental cells with endocytosed IONPs, endosomes, and EMs. The scale bars represent 500 nm. (B) Hydrodynamic diameters of BMSC-EM, BMSC-EV, 293T-EM, and 293T-EV are characterized by NTA. (C) Western blot analysis results of BMSC-EMs, BMSC-EVs, and BMSC cell lysates. <a href="https://www.jove.com/files/ftp_upload/66021/66021fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66021/66021fig04.jpg" /> 
Figure 4: The yield of EMs is higher than EVs. (A) The yield of EMs and EVs prepared from BMSC and 293T was analyzed by NTA. (B) Protein yield of EMs and EVs prepared from BMSC and 293T. **p &#60; 0.01. <a href="https://www.jove.com/files/ftp_upload/66021/66021fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66021/66021fig05.jpg" /> 
Figure 5: Representative fluorescent microscope images of PKH26-labeled EVs and EMs internalized by BMSC and ID8. The scale bars represent 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66021/66021fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles at JoVE.com

Author Spotlight: Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles 

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

Extracellular vesicles (EVs) have attracted significant attention in physiological and pathological research, disease diagnosis, and treatment; however, ...

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Biology

1.2K Views.  Tianjin University. Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs). 

Video: Author Spotlight: Development of a Large-Scale, Reproducible Production Method for Exosome Mimetics Using Magnetic Nanoparticles 

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

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