Isolation, purification, and characterization of EVs were performed by following MISEV 2018 Guidelines (19 (link)). Briefly, EDTA-blood was centrifuged 1,200 × g for 15 min at room temperature to obtain platelet-free blood plasma. Plasma was further centrifuged at 1,000, 2,000, and 3,000 × g for 15 min at 4°C, discarding the pellet to clean the cell debris. To prepare EV pellet for Nanosight and Flow Cytometry, 1.5 mL of fresh plasma was transferred into an ultracentrifuge tube (Quick-Seal®-Round-Top, Polypropylene, 13.5 mL-Beckman Coulter, Inc.) and filled up with PBS, filtered with 0.10 μm pore size membrane (StericupRVP, 0.10 μm, polyethersulfone filter- Merck Millipore) to minimalize the background contribution of interfering particles. Plasma was then ultracentrifuged (BeckmanCoulter Optima-MAX-XP) at 110,000 x g for 75 min at 4°C, to obtain an extracellular vesicles-rich pellet. The pellet was re-suspended with 500 μL triple 0.10 μm pore size membrane-filtered PBS. To prepare the EV pellet for miRNA extraction, 1.5 mL of fresh plasma was transferred into an ultracentrifuge tube (Centrifuge bottles polycarbonate, 10.4 mL-Beckman Coulter) and filled up with PBS. Plasma was then ultracentrifuged (BeckmanCoulter Optima-MAX-XP) at 110,000 x g for 75 min at 4°C, decanted, and the EV pellet was kept at −80°C until miRNA extraction.
Optima max xp
Manufactured by Beckman Coulter
Sourced in United States, Germany
The Optima MAX-XP is a high-performance ultracentrifuge designed for a wide range of applications in research and clinical laboratories. It features a compact design, advanced temperature control, and user-friendly software to ensure reliable and efficient sample processing.
Automatically generated - may contain errors
Lab products found in correlation
0.22 μm filter, by Merck Group (8 mentions)
Tla 55 rotor, by Beckman Coulter (5 mentions)
Rneasy cleanup kit, by Qiagen (4 mentions)
Nanosight ns300, by Malvern Panalytical (4 mentions)
Optima xpn 80, by Beckman Coulter (3 mentions)
Mls 50 rotor, by Beckman Coulter (3 mentions)
34 mm pc tubes, by Beckman Coulter (3 mentions)
Mla 130 rotor, by Beckman Coulter (3 mentions)
Stericuprvp, by Merck Group (2 mentions)
Tls 55 rotor, by Beckman Coulter (2 mentions)
Pkh26, by Merck Group (2 mentions)
Zetaview pmx 110, by Malvern Panalytical (2 mentions)
Ab212184, by Abcam (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Mirnaeasy kit, by Qiagen (2 mentions)
H 7650, by Hitachi (2 mentions)
3 mm glass bead, by Merck Group (2 mentions)
Mirneasy kit, by Qiagen (2 mentions)
Ca2 mg2, by Merck Group (2 mentions)
Optima xpn 100, by Beckman Coulter (2 mentions)
224 protocols using optima max xp
1
Extracellular Vesicles Isolation and Characterization
2
Extracellular Vesicle Isolation and Characterization
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EVs were isolated as described previously11 (link)19 (link)24 . Briefly, PFPs were diluted 1:1 in phosphate-buffered saline (PBS)/annexin binding buffer (ABB: 10 mmol/L HEPES, 140 mmol/L NaCl; 0.25 mmol/L CaCl2; pH: 7.4–7.5), followed by gravity-driven filtration through a 0.8 μm filter (Whatman). The filtrate was centrifuged at 20,500 g, 16 °C for 60 min in a microcentrifuge. The resulting MV pellet was resuspended in 50 μL buffer and was analyzed by flow cytometry (FCM). The supernatant was gravity filtered through a 0.2 μm filter (Sartorius), and then EXOs were pelleted by ultracentrifugation (UC) at 100,000 g, 4 °C, for 90 min (OptimaMAX-XP, MLA-55 rotor, Beckman Coulter Inc).
In some experiments, OptiprepTM-density UC was performed with both MV and EXO pellets as described previously42 (link). Briefly, 5, 10, 20 and 40 w/v% OptiprepTM (Sigma-Aldrich) layers were used starting with 40 w/v% at the bottom. The sample was layered onto the top, and UC was performed at 100,000 g, 4 °C for 20 h (Beckman Coulter OptimaMAX-XP, MLS-50 rotor). Fractions were collected from top to bottom. Each fraction was diluted with PBS and centrifuged at 100,000 g (MLA-55 rotor) or in the case of MVs, at 20,500 g. The pelleted fractions were either resuspended for FCM or lysed for Western blotting.
In some experiments, OptiprepTM-density UC was performed with both MV and EXO pellets as described previously42 (link). Briefly, 5, 10, 20 and 40 w/v% OptiprepTM (Sigma-Aldrich) layers were used starting with 40 w/v% at the bottom. The sample was layered onto the top, and UC was performed at 100,000 g, 4 °C for 20 h (Beckman Coulter OptimaMAX-XP, MLS-50 rotor). Fractions were collected from top to bottom. Each fraction was diluted with PBS and centrifuged at 100,000 g (MLA-55 rotor) or in the case of MVs, at 20,500 g. The pelleted fractions were either resuspended for FCM or lysed for Western blotting.
3
Talin Protein Stabilization using GraFix
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To stabilize weak protein-protein interactions, the GraFix crosslinking protocol was used (Stark, 2010 (link)). Talin samples were subjected to a sucrose and glutaraldehyde gradient of 10-30% and 0-0.2%, respectively, in a buffer containing 20 mM HEPES (pH 7.5), 75 mM KCl, 0.5 mM β-Mercaptoethanol and 0.5 mM EDTA. Gradients were prepared in 2.2 ml ultracentrifuge tubes (open-top polyclear tubes, Seton) using a Gradient Station machine (model ip, Biocomp). 50 μl of talin samples at a concentration of 5 mg/ml (approximately 18.5 μM) were placed on top of the gradients and centrifuged in a TLS55 rotor (Beckman Coulter) at 50 000 rpm, for 6 h at 4°C, using an Optima Max-XP ultracentrifuge (Beckman Coulter). 100 μl-Fractions were collected manually from top to bottom and quenched with 100 mM Tris (pH 7.5). Fractions containing the monomeric or dimeric talin, as determined by SDS-PAGE were pooled, concentrated, and buffer-exchanged to 20 mM HEPES (pH 7.5), 75 mM KCl, 0.5 mM β-Mercaptoethanol and 0.5 mM EDTA to remove excess sucrose using Amicon Ultra centrifugal filters (0.5 ml, 50 MWCO).
4
Quantifying α-Synuclein Fibrils by SDS-PAGE
5
Preparation of EV-depleted Fetal Bovine Serum
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EV-depleted FBS was prepared to avoid the presence of FBS-derived EV in EV preparations. FBS (Invitrogen) was ultracentrifuged by a 120.000× g for 16 h at 4 °C in a type MLA-55 rotor (Optima Max-XP, Beckman Coulter, Brea, CA, USA). The supernatant was aspirated cautiously and transferred to a new tube and then filtered through 0.22 μm filter (Millipore, Burlington, MA, USA) and was stored at −20 °C until use.
6
Isolation and characterization of P. gingivalis OMVs
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P. gingivalis cultures in late-exponential phase were used for OMV collection. A first centrifugation step at 8,000 × g and 4°C for 20 min was performed to separate cells from OMV-containing supernatant. The supernatant was subjected to ultracentrifugation at 100,000 × g and 4°C for 3 h in an Optima MAX-XP ultracentrifuge 261 (Beckman Coulter, Brea, CA, USA) using an MLA-80 fixed-angle rotor. The pellet containing the OMVs was resuspended in PBS, and aliquots were frozen at −80°C before use. Protein quantification was performed using a bicinchoninic acid (BCA) protein assay (Pierce, Waltham, MA, USA), according to the manufacturer’s instructions, with the addition of 2.0% SDS to solubilize proteins. Sixteen micrograms of protein was used for the phagocytosis rescue experiment. Protein precipitation with 10% tricarboxylic acid (TCA) was performed as described before (6 (link)) to concentrate vesicles for Western blot analysis. Recombinant PPAD was purified from Lactococcus lactis , as previously described (7 (link), 8 (link)).
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7
Isolation and Characterization of Macrophage Exosomes
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Exosomes were isolated from M0, M2 and GYY4137 pretreated-M2 macrophages. The macrophages were cultured in 1640 with exosome-free serum (centrifuged at 120,000g for 90 min) for 24h. The culture supernatant was collected and exosomes were isolated by ultracentrifugation according to previously described standard methods [47 (link)]. Optima MAX-XP (Beckman Coulter, USA) was used to perform ultracentrifugation experiments. The final exosomes were resuspended in PBS for further analysis. Using bicinchoninic acid to assay the protein amounts of exosomes. Transmission electron microscope (TEM, JEM-1400PLUS, Japan) was performed to observe exosomes. Nanoparticle tracking analysis (NTA) was performed at Shanghai Biotechnology Corporation (Shanghai, China) with the ZetaView PMX 110 (Malvern Instruments Ltd, UK) to quantify exosomes. PKH-26 (Sigma, Aldrich, USA) was used for exosome labeling according to the instructions of the manufacturer. For extraction of exosomes protein, the extracted exosomes were added with RIPA lysate containing 1% protease inhibitor and mixed evenly in EP tube. After standing at room temperature for 5 min min, intermixture was centrifuged at 12000g at 4 °C for 25 min min. Supernatant was transferred to a newly labeled EP tube.
8
EV Isolation from Platelet-Free Plasma
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Transport to the EPIGET Lab (University of Milan) and processing of blood samples was performed within two hours of collection. EDTA-blood was centrifuged at 1200× g for 15 min at room temperature to obtain platelet-free plasma. Plasma was further centrifuged at 1000, 2000, and 3000× g for 15 min at 4 °C, and the resulting pellets of cell debris were discarded. EV preparation and analyses were done in compliance with the MISEV 2018 guidelines (Detailed in Supplementary Table S1 ). To prepare EV pellets for Nanosight analysis and flow cytometry, 1.5 mL of fresh plasma was transferred to an ultracentrifuge tube (Quick-Seal, Round-Top, polypropylene, 13.5 mL; Beckman Coulter, Inc., Indianapolis, IN, USA), which was then filled up with phosphate-buffered saline (PBS) previously passed 3× through a sterile membrane filter unit (Stericup-VP, 0.10 µm, polyethersulfone; EMD Millipore, Billerica, MA, USA) in order to reduce background interferers as much as possible. Plasma was then spun in a benchtop ultracentrifuge (Optima MAX-XP; Beckman Coulter, Inc.) at 110,000× g for 75 min at 4 °C, to obtain an EV-rich pellet. The pellet was resuspended with 500 µL of triple membrane 0.1 µm filtered PBS for further analysis.
9
Isolation and Characterization of Extracellular Vesicles
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EVs were isolated by differential ultracentrifugation of the secretome of both WJ-MSCs and UC-MSCs (Optima MAX-XP, Beckman Coulter Inc., Irving, TX, USA), as previously described [32 (link)]. The size and concentration of pellet particles (n = 3 for each sample) were analyzed by NTA performed with a NanoSight (NS300, Malvern Instruments, Westborough, MA, USA) [62 (link)]. Briefly, five videos of each sample were recorded. Data analysis was performed with the NTA software and presented as mean ± standard deviation (SD) of the five video recordings. The resulting EV-depleted secretome was used in cell-based assays of tube formation, cell migration and proliferation.
10
Immunoprecipitation and Immunoblotting of α-Synuclein
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Flash frozen cortex was homogenised in 1% Triton‐X100 with Complete protease inhibitors. Protein concentration was quantified using a BCA assay. Homogenate containing 1 mg protein was ultracentrifuged at 120,000 g (Beckman Optima Max‐XP) for 1 h at 4°C. The pellet was dispersed in 1% Triton‐X100 using a needle to produce a homogenate containing a Triton‐insoluble fraction. From this homogenate, a 5% input was removed for subsequent immunoblotting. The remaining triton‐insoluble fraction was incubated overnight at 4°C with the IP antibody against α‐synuclein (1:100, Abcam ab212184). The mixture was then incubated with 25 μl Protein G Dynabeads according to the manufacturer's instruction. The protein of interest was eluted using NuPAGE LDS sample buffer and separated by electrophoresis on a 4%–12% polyacrylamide gel. Immunoblotting was performed using 5% milk blocking with overnight incubation at 4°C with an α‐synuclein antibody (1:1000, BD 610787). The membrane was incubated with a horseradish‐peroxidase‐conjugated anti‐mouse antibody (1:20,000, Sigma A9917) for 1 h, and visualised with chemiluminescence.
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