To purify the EVs, conditioned medium (CM) from CAFs and NFs was cleared by filtration through a 0.22‐mm PVDF filter (Millipore) and mixed with Total Exosome Isolation Reagent (from cell culture media) (Thermo Fisher Scientific, 4478359). EVs were isolated according to the manufacturer's instructions. The size and concentration of EVs were quantified using a NanoSight LM10V‐HS instrument (Malvern Instruments Ltd.) and transmission electron microscopy (TEM; Hitachi Hi‐Tech TH7700).11
0.22 mm pvdf filter
Manufactured by Merck Group
Sourced in United States
The 0.22-mm PVDF filter is a laboratory filtration device. It is designed to filter liquids and remove particles or contaminants as small as 0.22 millimeters in size.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick exosome precipitation solution, by System Biosciences (4 mentions)
Total exosome isolation reagent, by Thermo Fisher Scientific (2 mentions)
Exoquick tc exosome precipitation solution, by System Biosciences (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Zetaview particle tracker, by Particle Metrix (1 mentions)
Jem 1010, by JEOL (1 mentions)
5 protocols using 0.22 mm pvdf filter
1
Isolation and Characterization of Extracellular Vesicles
2
Exosome Isolation from Human Adipose Stem Cells
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hASCs were incubated for 1 day after seeding in a 100 mm dish (1 × 106 cells/dish), washed twice with PBS, and then cultured with 10 mL of DMEM without FBS for 24 h. After 24 h of incubation, the medium was collected and centrifuged at 3000 × g for 15 min at 4 °C, and then, only the supernatant was transferred to a new tube. The supernatant was filtered through a 0.22 mm PVDF filter (Millipore, Burlington, MA, USA). An appropriate volume of ExoQuick-TC™ Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) was added to the filtered culture medium and mixed well by inverting. After refrigeration for 12 h, the mixture was centrifuged at 1500×g for 30 min, and all supernatant was removed by aspiration. EV pellets were resuspended in 100 µL of PBS (phosphate buffered saline; Gibco) and stored at −80 °C for further use.
3
Exosome Isolation from Ovarian Cancer Cells
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Total exosome isolation reagent (Invitrogen) was used to isolate OC cells (HeyA8, HeyA8-MDR, SKOV3-ip1, SKOV3-TR, A2780, A2780-CP20), and cells were expanded in tissue culture plates. After cell cultures reached 70% confluency, cells were washed with PBS and incubated with 10% bovine exosome-depleted FBS (System Biosciences and/or Gibco) for 24 h. All cell culture media were collected in tubes and centrifuged at 2000 x g for 30 min, and the supernatant was filtered through a 0.22-mm PVDF filter (Millipore, Billerica, MA). An appropriate half volume of Total exosome isolation reagent (Invitrogen) was added to the filtered culture medium and incubated at 4 °C overnight. The samples were centrifuged at 10,000 x g at 4 °C for 1 h, and the exosome pellets were suspended in 150 μL PBS. Exosomes were then ready for RNA extraction or exosome quantification. Exosomes were diluted in PBS (1:100) and measured using a NanoSight instrument (Malvern, London, UK). Three replicate histograms for each sample were generated using the NanoSight instrument.
4
Exosome Isolation and Characterization
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The culture medium was collected and centrifuged at 3000×g for 15 min, and the supernatant was filtered through a 0.22-mm PVDF filter (Millipore). An appropriate volume of Exoquick exosome precipitation solution (System Biosciences) was added to the filtered culture medium, or a quarter volume of Exoquick exosome precipitation solution was added to the serum. After mixing and refrigeration for 24 h, the mixture was centrifuged at 1500×g for 30 min, and the supernatant was removed. Exosome pellets from 1 × 106 cells were suspended in 150 µL of serum-free medium. The size distribution and concentration of exosomes were analyzed by nanoparticle-tracking analysis using a ZetaView particle tracker from ParticleMetrix (Germany).
5
Exosome Isolation from AC16 Cells
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The collected culture media of AC16 cells were centrifuged at 3000 × g for 15 minutes, followed by filtering with a 0.22 mm PVDF filter (Millipore) to eliminate cellular debris. Then, exosomes were separated using the ExoQuick exosome precipitation solution (System Biosciences, CA, USA) as per the manufacturer's specifications. Transmission electron microscopy (TEM) (JEM-1010, JEOL, Tokyo, Japan) and nanoparticle-tracking analysis (NTA) were employed to identify exosome morphology, as well as size distribution and concentration.
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