EVs were isolated based on the protocol described previously (Qin et al., 2019 (link)). Briefly, cells were cultured in medium containing Exosome‐depleted FBS Media Supplement (SBI, USA). At 48 h after the preconditioning stimuli, the cell culture supernatant was collected and differentially centrifuged at 4°C for 10 min at 300 × g, then 15 min at 2000 × g and 45 min at 12,000 × g followed by filtration through a 0.22 μm PVDF filter (Millipore, USA). Finally, EVs were pelleted by ultracentrifugation at 120,000 × g for 70 min. The EVs were resolved in phosphate buffer saline (PBS) for the subsequent detection.
Pvdf filter
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Ireland
PVDF (Polyvinylidene fluoride) filter is a laboratory filtration product manufactured by Merck Group. It is designed to effectively separate and retain particles, molecules, or other materials from liquid samples. The filter's core function is to provide efficient filtration performance while maintaining the integrity of the filtered sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Polybrene, by Merck Group (5 mentions)
Pspax2, by Addgene (3 mentions)
Pmd2 g, by Addgene (3 mentions)
Exosome depleted fbs media supplement, by System Biosciences (3 mentions)
Exoquick reagent, by System Biosciences (3 mentions)
Ecl reagent, by GE Healthcare (3 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Plb vector, by Addgene (2 mentions)
Gel filtration standard, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
Ecl hrp detection reagents, by Cytiva (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Exoquick exosome precipitation solution, by System Biosciences (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Exoquick tc kit, by System Biosciences (2 mentions)
Fugene 6 transfection reagent, by Promega (2 mentions)
Nanoassemblr benchtop, by Precision Nanosystems (2 mentions)
50 kda mwco dialysis tubing, by Repligen (2 mentions)
115 protocols using pvdf filter
1
Extracellular Vesicle Isolation Protocol
2
CoQ10 Quantification in Plasma
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Blood was collected when mice were sacrificed at 16 months of age. For use, blood samples were allowed to thaw at 4 °C and centrifuged. Plasma levels of CoQ10 were determined according to methods described previously [29 (link),30 (link)]. Samples of plasma were deproteinated with methanol and n-hexane. This mixture was vortexed 5 min and then centrifuged at 2500× g for 20 min at 4 °C. Subsequently, the clear n-hexane layer was collected into another microcentrifuge tube, and the n-hexane extraction was repeated once more. Plasma extracts were evaporated to dryness using a stream of nitrogen gas. The dry residue was dissolved in mobile phase, filtered through a PVDF filter (4 mm, 0.45 μm; Millipore, Bedford, MA, and injected into the high-performance liquid chromatography (HPLC) system. A microBondapak C18 3.9 mm × 30-cm stainless steel column with a guard column 3 × 22 mm packed with microBondapak C18 was used with a mobile phase of methanol-n-hexane 85:15 (vol/vol). The flow rate was 1 mL/min, and the detector wave-length was 276 nm. The amounts of CoQ10 in plasma were identified according to a calibration curve that was constructed by plotting peak area vs. concentration. Linearity was achieved in the concentration range of 0.12 to 1.92 μg/mL.
3
Generating Peroxisome Knockout Cell Lines
4
Lentiviral Knockdown of Ask1 in RAW264.7 Cells
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Lentiviral plasmid vectors carrying Cas9 endonuclease, and sgRNA targeting Ask1 or carrying non‐targeting sgRNA, were prepared utilizing lentiCRISPRv2 plasmid (Addgene). The annealed oligos were cloned between the BsmBI restriction sites of the plasmid. The sequences of top and bottom oligos are listed in Table S1 . To produce lentivirus, HEK293T cells were transfected with Ask1‐targeting or non‐targeting lentiviral vectors, psPAX2 and pCMV‐SVS‐G (Addgene) plasmids using Lipofectamine 3000 (Thermo Fisher) in accordance with the manufacturer's protocols. To reduce cytotoxicity, the medium was replaced at 5 h after incubation. At 2 d after transfection, the supernatants containing lentivirus were harvested and filtered with 0.45 µm PVDF filter (Millipore). RAW264.7 cells were transfected with lentivirus and supplemented with 8 µg/mL polybrene (Nacalai Tesque). The medium was replaced at 1 d after transfection. Next day, the transfected cells were selected with 4 µg/mL puromycin (GIBCO).
5
Extracellular Vesicle Isolation from NPCE Cells
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EV samples were purified from NPCE cells under both normal (EVs-N) and oxidative stress conditions (EVS-OS) according to a PEG (Cat# 89510, Sigma, St. Louis, MO, USA)-based isolation method [40 (link),41 (link)] as previously described, with minor modifications. NPCE cells were plated at 5 million cells per 75 cm2 and after reaching 90% confluence, the cells were exposed for 1.5 h 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH)compound (Cat# 440914, Sigma, St. Louis, MO, USA). NPCE cell-conditioned medium was aspirated and centrifuged at 1500× g for 10 min to remove cells, followed by filtration through PVDF filter (0.22 µm, Millipore, Billerica, MA, USA) to remove large cellular debris. Precipitation solution (50% PEG8000, 0.5 M NaCl in PBS) was added to the cleared conditioned medium (1:5 v/v, respectively), mixed by flicking the tube and incubated overnight at 4 °C. After incubation, the tubes were centrifuged at 1500× g for 30 min at 4 °C to acquire the pellet of EVs. The supernatant was discarded and pelleted EVs were dissolved in 500 µL PBS for further analysis. The EVs-N were isolated from non-treated (NT) NPCE cells following the same procedure as described above.
6
Isolation of Exosomes from Mouse Granulosa Cells
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Mouse granulosa cells were acclimated in DMEM/F12 without exosomes for 24 h, and then subjected to different interventions. No special treatment was provided to the control group (CON). The ferric citrate (FAC) group was treated with 100 μM ferric citrate. The deferoxamine mesylate (DFO) group was treated with ferric citrate (100 μM) and deferoxamine mesylate (100 μM). VE group was treated with ferric citrate (100 μM) and VITE (200 μM). All groups were cultured for 48 h. After the termination of incubation, centrifugation was carried out in steps at 4 °C and 300 × g for 10 min to remove cells and 3000 × g for 10 min to remove large cell debris. After centrifugation at 10,000 × g for 40 min, the supernatant was filtered through a 0.22 μM PVDF filter (Millipore, USA). Finally, centrifugation at 100,000 × g for 90 min, the supernatant was removed and resuspended in 200 μL of PBS to obtain the cell supernatant exosomes precipitation.
7
Extracellular Vesicle Isolation from Ascites
8
HPLC Quantification of Two Standards
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Concentrated solutions of two standards (3 and 4 ) were prepared in the ACN, each at 1000 mg/L. After diluting the stock solutions with ACN to obtain the developing solutions, seven concentration levels were found in the range of 2.5–500 mg/L. The worked solutions were filtered by a PVDF filter (0.45 µM, Millipore) before HPLC injection. Linear regression analysis was applied to achieve linearity by the integrated peak areas (Y) vs. the concentration of each standard (X, mg/L) at five different concentrations.
Analysis conditions: 35% to 40% B from 0 to 15 min, 40% to 60% B from 15 to 20 min, 60% B from 20 to 25 min, 60% to 70% B from 25 to 35 min, and 70% to 80% B from 35 to 60 min.
Analysis conditions: 35% to 40% B from 0 to 15 min, 40% to 60% B from 15 to 20 min, 60% B from 20 to 25 min, 60% to 70% B from 25 to 35 min, and 70% to 80% B from 35 to 60 min.
9
Extraction of Acerola Juice Extracellular Vesicles
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Acerola juice was obtained from Nichirei Biosciences (Saitama, Japan). The ELNs derived from acerola juice were extracted using an exoEasy maxi kit (QIAGEN, Valencia, CA, USA) and ExoQuick reagent (System Biosciences, Mountain View, CA, USA), in accordance with the manufacturers’ protocols.
In the methods using an exoEasy maxi kit, 8 mL of acerola juice was filtered through a 0.45-μm polyvinylidene fluoride (PVDF) filter (Millipore, Billerica, MA, USA), supplemented with an equal volume of buffer XBP, mixed well, transferred into an exoEasy spin column, and subjected to treatment in line with the manufacturer’s instructions. A total of 800 μL of buffer XE was added, followed by centrifugation for 5 min at 5,000 × g, after which the flowthrough was concentrated by ultracentrifugation at 100,000 × g using a TLA-110 rotor 1 (Beckman Coulter, Brea, CA, USA) for 70 min at 4°C to change the elution buffer XE to 30 μL of phosphate-buffered saline (PBS).
In the methods using ExoQuick reagent, 8 mL of acerola juice was filtered using a 0.45-μm PVDF filter, and 2 mL of ExoQuick reagent was added and mixed thoroughly by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1,500 × g for 30 min and all supernatant was removed by aspiration, after which 30 μL of PBS was added for resuspension.
In the methods using an exoEasy maxi kit, 8 mL of acerola juice was filtered through a 0.45-μm polyvinylidene fluoride (PVDF) filter (Millipore, Billerica, MA, USA), supplemented with an equal volume of buffer XBP, mixed well, transferred into an exoEasy spin column, and subjected to treatment in line with the manufacturer’s instructions. A total of 800 μL of buffer XE was added, followed by centrifugation for 5 min at 5,000 × g, after which the flowthrough was concentrated by ultracentrifugation at 100,000 × g using a TLA-110 rotor 1 (Beckman Coulter, Brea, CA, USA) for 70 min at 4°C to change the elution buffer XE to 30 μL of phosphate-buffered saline (PBS).
In the methods using ExoQuick reagent, 8 mL of acerola juice was filtered using a 0.45-μm PVDF filter, and 2 mL of ExoQuick reagent was added and mixed thoroughly by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1,500 × g for 30 min and all supernatant was removed by aspiration, after which 30 μL of PBS was added for resuspension.
10
Lentiviral Transduction and Genome Editing
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