Isolated exosomes were characterized by qNano-IZON (Izon, Cambridge, MA, USA) by measuring the concentration (particles/mL), size-distribution, and particle diameter. The tunable resistive pulse sensing technology (qNano-IZON system) allows exosome detection by driving vesicles through a pore using a combination of electrophoretic and convective flow induced by applied voltage and external pressure across the pore, respectively (Datta et al. [37 (link)]). Initially, we calibrated the voltage, stretch, pressure, and baseline current using two standard beads: CPC100 (mode diameter, 115 nm [Izon] and a concentration of 1.1 × 1013 particles/mL) and CPC70 (mode diameter of 70 nm [Izon] and a concentration of 1.9 × 1013 particles/mL). Finally, we optimized the system at a stretch of 47.99 mm, with a voltage of 0.58 V and a pressure level of 5.0 mbar. For analyses, 40 μL of diluted sample was placed in the upper fluid cell under identical conditions. The upper fluid cell was washed with PBS to remove residual particles after each application to prevent cross-contamination. NP100 pore type is applicable to the intended measurement for 50–200 nm size range of the extracellular vesicles. For analysis with NP100, the samples were filtered by 0.45-μm Millex-HV Syringe Filter (EMD Millipore). The data analysis was performed with the qNano-IZON software.
Millex hv syringe filter
Manufactured by Merck Group
Sourced in United States, Ireland
The MILLEX®-HV syringe filter is a sterile, disposable filtration device designed for the rapid filtration of small sample volumes. It features a high-performance hydrophilic membrane that effectively removes particulates and microorganisms from a variety of aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Toc 5 analyser, by Shimadzu (1 mentions)
Avanti 30 centrifuge, by Beckman Coulter (1 mentions)
Strata x 33 m polymeric reversed phase cartridges, by Phenomenex (1 mentions)
Lentiviral packaging mix, by Thermo Fisher Scientific (1 mentions)
Viralpower lentiviral expression system, by Thermo Fisher Scientific (1 mentions)
Plenti6 v5 gw lacz, by Thermo Fisher Scientific (1 mentions)
15 protocols using millex hv syringe filter
1
Exosome Characterization via Tunable Resistive Pulse Sensing
2
Water Quality Measurement Techniques
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Water temperature was measured using a glass thermometer (Fisher Scientific Ltd, Loughborough, UK). Turbidity measurements were performed using a HACH model 2100N turbidity meter (Camlab, Cambridge, UK). The equipment was calibrated daily, and results were within 95 % accuracy. Water pH was measured using a VWR pH meter (VWR International Ltd., Leicestershire, UK). The equipment was calibrated before each batch of analysis. For DOC and SRP measurements, samples were filtered through a 0.45-μm Millex-HV syringe filter (Merck Millipore, Tullagreen, Ireland) prior to analysis. DOC analysis was carried out using a Shimadzu TOC-V analyser (Shimadzu, Tokyo, Japan). The equipment was calibrated with 100 mg L−1 standard solutions before each use and, in all cases, 100 % accuracy in the measurements was obtained. SRP measurements were carried out using Merck Millipore orthophosphate test kits with a measurement range of 0.05–5 mg L−1 using a Merck Spectroquant NOVA 60 spectrophotometer (Merck Millipore, Tullagreen, Ireland). The photometer was calibrated weekly, and analyses were performed in duplicates.
3
Extraction of Phenolic Compounds
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1 g of leaves were homogenized with 10 ml of 80% methanol containing 1% acetic acid and filtered through a 0.45 μm filter using a MILLEX®-HV syringe filter (Millipore Corporation, Bedford, MA, USA) by the method of Khanam et al. [33 (link)]. The filtrate was centrifuged at 10,000 g for 15 min and used to analyze phenolic acids and flavonoids.
4
Isolation and Lyophilization of Mohave Rattlesnake Venom
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Lyophilized crude venom from C. s. scutulatus (Mohave rattlesnake, Type A) was obtained from an individual adult snake housed in the National Natural Toxins Research Center (NNTRC) serpentarium, located at Texas A&M University-Kingsville, Kingsville, TX. Venom extraction was performed by placing disposable plastic cups covered with parafilm in close proximity to the snakes. The extracted venom was subsequently centrifuged at 10,000× g at 4 °C for 5 min using a Beckman Coulter Avanti 30 Centrifuge. The venom was then filtered through a 0.45 µm MillexHV syringe filter unit (Millipore, Billerica, MA, USA), followed by lyophilization. Finally, the lyophilized venoms were stored at −80 °C until further use. Css-CRiSP was purified as described by Suntravat et al. [12 (link)].
5
Extraction of Phenolic Compounds from Leaves
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One gram of fresh-frozen leaves was homogenized with 10 ml of 80% methanol containing 1% acetic acid. The homogenized mixture was filtered through a 0.45 µm filter using a MILLEX®-HV syringe filter (Millipore Corporation, Bedford, MA, USA) and centrifuged at 10,000 g for 15 min. The final filtrate was used to analyze phenolic acids and flavonoids.
6
Betacyanin Extraction from Plant Leaves
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10 mL of 80% methanol containing 1% acetic acid was added in 1 g of leaves and homogenized thoroughly, and transferred to a 50 mL tightly capped test tube. The test tubes were placed in a shaker (Scientific Industries Inc., USA) for 15 h at 400 rpm. 0.45 µm filter (MILLEX-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. The mixture was centrifuged at 10,000 × g for 15 min. Betacyanin components were analyzed from the final filtrate. Betacyanin analysis in the samples could interfere through the precipitation of methanol with the proteins and other insoluble substances in the samples. Strata-X 33 µm Polymeric Reversed-Phase cartridges (Phenomenex, Torrance, CA, USA) were used to purify betacyanin. All extractions were done in triplicate independent samples.
7
Flavonoid Extraction from Leaf Samples
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The fresh leaf samples were extracted by adding 10 ml methanol (80%) containing acetic acid (1%) in 1 g leaves. The mixture was thoroughly homogenized. Then the mixture was kept in a test tube (50 ml) and capped tightly. The test tube was shaken in a shaker (Scientific Industries Inc., USA) for 15 h at 400 rpm. An exactly 0.45 μm filter (MILLEX®-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. We centrifuged the mixture at 10,000 × g for 15 min. The flavonols, flavanols, flavones, and flavanones were analyzed from the final filtrate. We performed all extractions in triplicate independent samples.
8
Lentiviral Expression of NOX4 Protein
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The NOX4/pCMV-SPORT6 vector was obtained from 21C Frontier Human Gene Bank (KRIBB, Daejeon, Korea). Lentivirus was produced using the Viralpower Lentiviral Expression System (Invitrogen). Briefly, the NOX4 open reading frame (ORF) was amplified from the NOX4/pCMV-SPORT6 vector using the following primers: forward (5′-CCCGGGACCATGGCTGTGTCC-3′) and reverse (5′-GCGGCCGCTCAGCTGAAAGACTC-3′). The NOX4 cDNA was subcloned into the pLenti6/V5-D-TOPO vector according to the manufacturer's protocol. The resultant NOX4/pLenti6-D-TOPO vector was verified using PCR, restriction digestion, and sequence analysis (Macrogen, Seoul, Korea). The pLenti6/V5-GW/lacZ (Invitrogen) vector was used as a positive control. Recombinant lentiviruses were produced according to the manufacturer's protocol (Invitrogen). Briefly, we co-transfected the pLenti6/V5-D-TOPO (NOX4 or LacZ) vector and the Lentiviral Packaging Mix (Invitrogen) into 293 FT cells. After 48 h, the viral supernatant was collected, spun to remove cell debris, filtered through a Millex-HV syringe filter (0.45 μm, Millipore, Bedford, MA, USA), and stored at −80 °C.
9
Extraction of Flavonoids and Phenolic Acids
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10 ml of 80% methanol containing 1% acetic acid was added in 1 g of fresh leaves and homogenized thoroughly. 0.45 µm filter (MILLEX®-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. The mixture was centrifuged at 10,000 × g for 15 min. Flavonoids and phenolic acids were analyzed from the final filtrate.
10
Fecal Sample Processing Protocol
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Collected samples on ESwab™ buffer were defrosted and homogenized for 2 min by vortexing, then centrifuged at 4,000 rpm for 10 min in Eppendorf tubes. The supernatant was recovered in a new Eppendorf tube and stored at −20°C until use. Further processing by filtering was done by using a 13 mm diameter sterile syringe filter with a 0.45 µm or 0.2 µm pore size hydrophilic PVDF membrane (Millex-HV Syringe Filter, Millipore). The dilution of feces solution was done following the general mixing of volumes; 75 µl 2X LB medium, plus 1.5 µl of biosensor culture, plus 3 µl of inducer adjusted at the needed concentration and 70.5 µl of the feces solution diluted in ESwab™ buffer to have a final volume of 150 μl, as follow; for 10% final feces concentration: 15 µl of feces samples plus 55.5 µl of ESwab™ buffer, for 25% final feces concentration: 37.5 µl of feces samples plus 33 µl of ESwab™ buffer and for 50% dilution 75 µl of feces samples. See Supplementary Protocols for a step-by-step procedure.
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