HepDE19 cells16 (link) (generously provided by Dr. Haitao Guo, Indiana University) were cultured on collagen-coated T175 flasks in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 3% FBS and 0.1 mM non-essential amino acids (NEAA). The cells were cultured in the absence of tetracycline to induce HBV replication. After seven days in culture without tetracycline to induce HBV expression, supernatant was collected every other day for two weeks and fresh medium was added. After each collection, medium was spun down at 1,000 × g at 4 °C to remove any cell debris and passed through a 0.22 μm filter. During this process, the medium was kept at 4 °C. At the end of all the collections the medium was concentrated 100-fold via centrifugation using Centricon Plus-70 centrifugal filter devices (Millipore-Sigma, Billerica, MA). The concentrated virus stock was aliquoted and stored at −80 °C. One of the aliquots was used to determine the HBV genomes/ml with a TaqMan-based qPCR method.
Centricon plus 70
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
The Centricon Plus-70 is a laboratory centrifugal device used for sample concentration and purification. It features a high-performance membrane that allows for the efficient separation and concentration of molecules, proteins, and other biological components from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 15, by Merck Group (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
0.22 μm pore filter, by Merck Group (2 mentions)
Whatman, by Cytiva (2 mentions)
Sw55ti rotor, by Beckman Coulter (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
293fectin, by Thermo Fisher Scientific (2 mentions)
Biomax 100, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Serum free neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Hregf, by Thermo Fisher Scientific (2 mentions)
Hrbfgf, by Thermo Fisher Scientific (2 mentions)
Biomerieux nuclisens kit, by bioMérieux (2 mentions)
Kingfisher ml, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (2 mentions)
Lenti x 293t cells, by Takara Bio (2 mentions)
Beckman optima xl a, by Beckman Coulter (2 mentions)
Optima le 80k ultracentrifuge, by Beckman Coulter (1 mentions)
97 protocols using centricon plus 70
1
HBV Replication in HepDE19 Cells
2
Isolation and Characterization of Extracellular Vesicles
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Isolation and characterization of EVs was in accordance with the minimum information for studies of extracellular vesicles (MISEV) published in 2018 [42 (link)], as summarized by ISEV. EVs are characterized by their expression of vesicle-associated proteins (CD63, CD81, ITGAV) and diameter range of 40–150 nm. In this study, stromal cells (HCKs, HCFs, or HCMs) were cultured for 36 h in serum-free media, the conditioned media (CM) was collected, and EVs were isolated, as previously described [11 (link)]. In brief, stromal cell CM underwent serial differential centrifugation to remove cells (300× g for 10 min), cellular debris (3000× g, for 10 min), and apoptotic detritus (13,000× g for 30 min). The supernatant was concentrated using a Centricon® Plus-70 centrifugal filter unit with a 100 kDa MW cutoff (MilliporeSigma, Burlington, MA, USA), and ultracentrifuged for 1 h and 10 min at 110,000× g (4 °C) using a Beckman Type 50.2 Ti Rotor (Beckman Coulter, Brea, CA, USA) in a Beckman Coulter, Optima LE-80K Ultracentrifuge. The resultant pellet was resuspended in phosphate buffered saline (PBS; Gibco), centrifuged again for 1 h and 10 min at 110,000× g (4 °C), and stored at −80 °C.
3
Purification of PACE4 Recombinant Protein
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For each purification, 100–150 mL of conditioned medium of Schneider 2 cells, stably expressing construct encoding hPACE4-FL and hPACE4-altCT cDNA C-terminally tagged with 6xHis-V5 in pAC5.1 vector, were buffer-exchanged and concentrated on 30 kDa molecular filtering centrifugal devices (Centricon Plus-70, Millipore Sigma) and purified on a nickel chelating-resin (see Supplementary methods ). PACE4 concentration in recombinant protein preparations were determined by quantitative LC–MS/MS methods monitoring PACE4 tryptic peptides (see Supplementary methods ).
4
Isolation and Proteomic Profiling of EVs
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We plated an equal number of mFT cells (mFT3635, mFT3666) in a 150 cm2 culture dish. When cells reached about 99% confluency, they were rinsed with PBS and allowed to grow in a serum‐depleted culture medium (48 h). We next collected cell culture media and removed cell debris via centrifugation (10 000×g, 3 min). To collect EVs, we first concentrated supernatants using a centrifugal filter (Centricon Plus‐70, Millipore Sigma) and loaded a concentrated media (0.5 mL) to a qEV column (IZON, SP1). EV fractions (F7‐F9) were collected (1.5 mL). Total protein concentrations were measured via a Qubit protein assay (ThermoFisher, Q33212). For proteome profiling, isolated EVs were sent to an external vendor (BGI company) that performed EV lysis, protein recovery, sample digestion, and nano‐flow LC‐MS/MS analysis. Abundant proteins were selected by the spectral counting method provided by BGI.
5
Exosome Isolation from Conditioned Media
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293F cells were grown in Freestyle media as described above for a period of 3 days. Cells were removed by centrifugation at 300g for 5 min, and large cell debris was removed by centrifugation at 3000g for 15 min. This generated the conditioned media that was passed through an ∼200 nm pore size diameter sterile filtration unit to generate a CTCS. The CTCS was concentrated by centrifugal filtration (Centricon Plus-70, MilliporeSigma), with ∼120 ml CTCS yielding a concentrated vesicle supernatant (CVS) of ∼0.5 ml. Vesicles in the CVS were then separated from free protein by SEC, using PBS as column buffer (qEV column, Izon). Exosomes eluting in the postvoid fractions and the three peak exosome fractions were pooled. This pool of peak exosome-containing fractions was purified further by another round of centrifugal filtration (Ultra-4, 100 kDa cutoff, Amicon), generating the purified exosome preparation.
6
Isolation of Extracellular Vesicles from Cell Culture Media
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CCM was thawed and concentrated in preparation for EV isolation by size-exclusion chromatography followed by additional concentration of the EV sample. All three steps were performed according to the manufacturer’s instructions. Briefly, Centricon® Plus-70 centrifugal filter units (10,000 kDa cutoff, MilliporeSigma) were primed with 50 mL of 0.1 N sodium hydroxide, followed by 65 mL of 0.22 µm-filtered PBS. The retentates of the priming buffers were discarded. Then, 50 mL portions of CCM were concentrated to 500 µL, which was loaded into a SEC column (qEVoriginal, 70 nm, IZON Science) that had been primed with 13 mL of 0.22 µm-filtered PBS. SEC-output fractions of 500 µL each were collected. The fractions with highest abundance of EVs but with the least protein contaminants are fractions 7 to 10 according to the product manual and our previous findings [27 ]. For each replicate, the EV fractions (i.e., 7 to 10) of five SEC column isolations were combined and concentrated from 10 mL to 200 µL. Of this final EV-product for each replicate, 2 µL was used for counting and TEM. The remainder was used for RNA extraction (Fig. S1b).
7
Exosome Isolation and Fluorescent Labeling
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The medium of both M0- (non-induced control) and M1-like macrophages was replaced with fresh D˗MEM, supplemented with 10% v/v exosome-depleted FBS for 48 h, before culture-conditioned medium (CCM) collection. The collected CCM was previously centrifuged twice at 700 × g for 5 min and 2000 × g for 10 min at 4 °C and then filtered through a 0.22 μm filter unit, in order to remove debris and detached cells [26 ]. Later, the starting volume was concentrated by using Centricon Plus-70 centrifugal filters (Millipore Sigma) up to a final volume of 1 mL. The samples’ purification was performed through size exclusion chromatography (SEC) with qEV original 70 nm columns (Izon Science, Cambridge, MA). All the columns were rinsed with PBS, and then 1 mL of each sample was loaded onto each column, followed by fractions’ extraction. Finally, the recovered volume was concentrated by using 50 kDa MWCO Amicon Ultra-15 centrifugal filters up to a final volume of 1 mL [27 (link)]. When required, fluorescent EVs were labeled after isolation by 1 h of incubation at 37 °C with carboxyfluorescin diacetate succinimidyl ester (CFDA-SE). This latest, after passing the membranes, undergoes hydrolysis of the diacetic groups, thus coverting into the fluorescent state CFSE [28 (link), 29 (link)]. The fluorophore excess was removed by the use of centrifugal filters.
8
Isolation and Characterization of ARPE-19 Extracellular Vesicles
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Cell culture media (serum-free) from ARPE-19 was filtered through 0.22-um filter and concentrated with Centricon Plus 70 (100K NMWL, MilliporeSigma). EVs for proteomic study were isolated using a sucrose cushion and iodixanol buoyant density gradient ultracentrifugation (Dong-Sic Choi, 2016; Zhou et al., 2020c) . Briefly, the concentrated cell culture media was added to the 0.8 M sucrose and 2 M sucrose cushion. After ultracentrifugation, EVs were located at the interface between two sucrose cushion buffer layers. The interface solution was collected and placed at the bottom of an iodixanol buffer. After ultracentrifugation, 10 fractions remained in the tube, with EVs in the third fraction. The 10 fractions were collected and ultracentrifugation was conducted on each. The 10 pellets were re-suspended in 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES)-Buffered Saline (HBS).
EVs for characterization and western blot studies were isolated using differential centrifugation.
Briefly, cell culture media was filtered using a 0.22-µm filter and then was concentrated to 1/10th of its original volume using Amicon Centrifugal Filter Ultra-15mL 3K. Concentrated cell culture media was ultracentrifuged at 100,000 x g for 17 h and was washed using HBS at 100,000 x g for 5 h. EV pellets were re-suspended in HBS buffer.
EVs for characterization and western blot studies were isolated using differential centrifugation.
Briefly, cell culture media was filtered using a 0.22-µm filter and then was concentrated to 1/10th of its original volume using Amicon Centrifugal Filter Ultra-15mL 3K. Concentrated cell culture media was ultracentrifuged at 100,000 x g for 17 h and was washed using HBS at 100,000 x g for 5 h. EV pellets were re-suspended in HBS buffer.
9
Concentration of HBV for In Vivo Studies
10
Wastewater Concentration Methods Comparison
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Untreated and treated wastewater samples were concentrated using three different techniques: ultrafiltration [7 (link)]; adsorption-elution [11 (link)]; and adsorption-extraction [20 (link)]. Remaining samples from April to July were processed using the adsorption-extraction method due to a shortage of supplies related to other methods. Ultrafiltration (Method A) applied the Centricon® Plus-70 centrifugal filter with a nominal molecular weight limit (NMWL) of 100 kDa (Merck Millipore; part no UFC710008, Burlington, MA, USA). The adsorption-elution method (Method B) used an electronegative membrane as described elsewhere [3 (link),21 (link)]. The adsorption-extraction method (Method C) also used an electronegative membrane, as well as a wastewater sample amended with MgCl2 pretreatment as reported previously [20 (link)].
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