Centricon plus 70 centrifugal filter

Manufactured by Merck Group

Sourced in United States

The Centricon Plus‐70 Centrifugal Filter is a laboratory instrument used for the concentration and purification of macromolecules, such as proteins and nucleic acids, from complex solutions. The device utilizes centrifugal force to separate the desired molecules from the surrounding fluid, allowing for efficient sample preparation and recovery.

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Lab products found in correlation

Exosome depleted fbs, by Thermo Fisher Scientific (2 mentions) Nanosight ns300, by Malvern Panalytical (2 mentions) Amicon ultra 4 centrifugal filter unit, by Merck Group (2 mentions) Qev original 70 nm column, by Izon Science (2 mentions) Amicon ultra 2 centrifugal filters, by Merck Group (2 mentions) Exoquick exosome precipitation solution, by System Biosciences (2 mentions) Qubit assay kit, by Thermo Fisher Scientific (1 mentions) Tla45 rotor, by Beckman Coulter (1 mentions) Amicon ultra 15, by Merck Group (1 mentions) Microbca, by Thermo Fisher Scientific (1 mentions) Dc assay, by Bio-Rad (1 mentions) Brilliant violet 605, by BD (1 mentions) Benzonase, by Merck Group (1 mentions) Millex gp, by Merck Group (1 mentions) Amicon ultra 4 10 kda nominal, by Merck Group (1 mentions) 0.22 μm filter, by Merck Group (1 mentions) Qev size exclusion chromatography column, by Izon Science (1 mentions) Qev sec columns, by Izon Science (1 mentions) 0.22 μm filter, by Celltreat (1 mentions) Cariporide, by Merck Group (1 mentions)

Close spelling variants of this product

Centricon Plus-70 100-kDa Centrifugal Filter Devices Centricon® Plus-70 Centrifugal Filter Units Centricon Plus-70 filter Centricon Centrifugal filter Centricon Plus-70 Centricon filters Centrifugal filter Centricon

18 protocols using centricon plus 70 centrifugal filter

Characterization of Extracellular Vesicles

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EVs preparation, isolation, and characterization. CDCs were conditioned in Iscove's Modified Dulbecco's Medium (IMDM) without supplementation for 15 days at 37°C and 20% O2. The conditioned media was collected, filtered through a 0.45 μm filter, and concentrated using 10 KDa Centricon Plus‐70 Centrifugal Filter (Millipore) or 1000 KDa Centricon Plus‐70 Centrifugal Filter (Millipore). Protein concentration was measured using the DC assay (Biorad). Particle size and concentration were measured on a NanoSight NS300 (Malvern). The parameters for Nanosight acquisition and analysis were as follows:
Camera level: 15
Detection Threshold: 5
Number of videos acquired per sample: 4
Video duration: 30 s

Ciullo A., Li C., Li L., Ungerleider K.C., Peck K., Marbán E, & Ibrahim A.G. (2022). Biodistribution of unmodified cardiosphere‐derived cell extracellular vesicles using single RNA tracing. Journal of Extracellular Vesicles, 11(1), e12178.

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Extracellular Vesicle Harvesting and Purification

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In order to test different antibodies and experimental conditions prior to clinical use, we harvested EV from the culture of cell lines listed above. Cells were grown for 48-72 h in normal growth medium supplemented with 5% exosome-depleted FBS (Thermo, A2720801). Conditioned media was collected and centrifuged at 300 × g for 10 min to remove dead cells and debris, followed by filtration through a 0.22-μm cellulose acetate vacuum filter (Corning, 430767). Media was then concentrated to ~ 1 mL using a Centricon Plus-70 Centrifugal Filter (Millipore Sigma, UFC710008) and centrifugation at 4000 × g for 20 min, following the protocol of Lobb et al.^{33 (link)}. EV were then purified from the concentrated media using a qEV original column from IZON (iZON Science, SP1) following the manufacturer’s instructions.

Yang K.S., O’Shea A., Zelga P., Liss A.S., Del Castillo C.F, & Weissleder R. (2023). Extracellular vesicle analysis of plasma allows differential diagnosis of atypical pancreatic serous cystadenoma. Scientific Reports, 13, 10969.

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Isolation and Characterization of Extracellular Vesicles

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Cells were incubated in a medium with 2% exosome‐depleted FBS (Thermo Fisher) for 48 h, followed by EV collection. The conditioned medium was collected through a cell strainer (40 µm Nylon, Thermo Fisher) and filtered through a 0.2 µm membrane filter (Millipore Sigma). The conditioned medium was concentrated with Centricon Plus‐70 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma) and centrifuged at 3,500 g for 30 min at 4 °C. The concentrated medium was passed through with SEC. Similar to EV isolation from bile samples, the 4th and 5th fractions were used for EV isolation, followed by concentration using Amicon Ultra‐2 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma) and centrifuged at 3500 x g for 30 min at 4 °C. The isolated EVs were reconstituted in PBS, aliquoted, and stored in a −80 °C deep freezer. Total EV protein was measured using a Qubit assay kit (ThermoFisher, Q33212). The isolated EVs were characterized by transmission microscopy, western blot, and nanoparticle tracking analysis (Figure S17, Supporting Information).

Jeong M.H., Son T., Tae Y.K., Park C.H., Lee H.S., Chung M.J., Park J.Y., Castro C.M., Weissleder R., Jo J.H., Bang S, & Im H. (2023). Plasmon‐Enhanced Single Extracellular Vesicle Analysis for Cholangiocarcinoma Diagnosis. Advanced Science, 10(8), 2205148.

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Cytokine Profiling of Conditioned Media

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Conditioned media from KCs was concentrated 5× by centrifugation through a Centricon Plus-70 Centrifugal Filter with a cutoff of 3 kDa (MilliporeSigma), according to the manufacturer’s instructions. Protein concentration was normalized between paired samples, and a 1 ml sample was incubated, according to the manufacturer’s instructions, on a Human Cytokine C3 Array (RayBiotech). Dot blot images were digitized using an HP OfficeJet 5,610 All-in-One office scanner at 300 dpi and saved as grayscale mode TIFF documents. Densitometry was performed as described in Johnson et al. (2016) (link) Densitometry values for each analyte were normalized to within-membrane-positive and within-membrane-negative control dots, and Dsg1 membrane dot values were normalized to the macrophage-derived chemokine (MDC) dots on the paired control membrane since this secreted factor was detectable on every membrane and had a low level of experiment to experiment variability.

Arnette C.R., Roth-Carter Q.R., Koetsier J.L., Broussard J.A., Burks H.E., Cheng K., Amadi C., Gerami P., Johnson J.L, & Green K.J. (2019). Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling. Pigment cell & melanoma research, 33(2), 305-317.

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Exosome Isolation and Characterization

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Cells were incubated at 37 °C in 5% CO₂ and were cultured in T-75 flasks until they reached around 80–90% confluency. Cells were then washed three times with PBS and incubated in the exosome-harvesting medium for 48 h. Exosome-harvesting medium includes DMEM plus 0.5% exosome-depleted FBS, as described previously [24 (link)]. The conditioned medium (CM) was harvested and centrifuged at 2000× g for 20 min at 4 °C to remove cells, apoptotic bodies, and cell debris. The medium was then concentrated around 10 times via Centricon^® Plus-70 centrifugal filter with a 10 K molecular weight limit (Millipore, MA, USA) under 3500× g for 20 min. An invert spin was then performed at 1000× g for 2 min at 4 °C. The concentrated media was added to a qEV/original SEC (Izon Science); the fragment between 3–5.5 mL was collected and analyzed.

Song Z., Mao J., Barrero R.A., Wang P., Zhang F, & Wang T. (2020). Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation. Molecules, 25(23), 5585.

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Isolation and Characterization of Extracellular Vesicles

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Cell lines were cultured for 24 h without serum before EVs isolation. The following day concentrated conditioned medium (CCM) was harvested by pelleting cells at 400 × g for 10 min at 4 °C. Supernatant was centrifuged at 2,000 × g for 20 min at 4 °C to discard 2K pellet and then concentrated on a sterilized Sartorius Centrifugal Filter (MWCO = 100 kDa; VS2061) or Centricon Plus-70 Centrifugal Filter (MWCO = 100 kDa; Millipore). Medium was concentrated to 500 μL and overlaid on 70 nm qEV size-exclusion columns (Izon, SP1) for separation. Twenty-two 500-μL fractions were recovered and analyzed separately or pooled, 7–10 as EV-Rich (EV-R) fraction and 15–22 as EV-Poor (EV-P) fraction. Pooled fractions were then concentrated using 10 KDa cutoff filters (Amicon Ultra-15, Millipore). Protein concentration in EV-R, EV-P, or CCM was measured using Micro-BCA (Thermo Scientific) in the presence of 0.2% SDS.
A total of 200–400 µL of thawed human patients’ tumor supernatants were ultracentrifuged at 100,000 × g for 30 min in a TLA-45 rotor (Beckman Coulter). Pellets were resuspended in 25 µL of PBS.

Tkach M., Thalmensi J., Timperi E., Gueguen P., Névo N., Grisard E., Sirven P., Cocozza F., Gouronnec A., Martin-Jaular L., Jouve M., Delisle F., Manel N., Rookhuizen D.C., Guerin C.L., Soumelis V., Romano E., Segura E, & Théry C. (2022). Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome. Proceedings of the National Academy of Sciences of the United States of America, 119(17), e2107394119.

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Generation of Tetramer Probes for Flow Cytometry

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For tetramer probe generation, rPfCSP or S02-DsbC (S02 fused to the disulfide bond C protein) were first biotinylated and then respectively conjugated to the fluorophores FITC (fluorescein isothiocyanate) and BV605 (Brilliant™ Violet 605) (BD Biosciences). Biotinylation was performed using ligase Bir A (Avidity) at 30°C for 4 hours prior to buffer exchange with 1X PBS (pH 7.4) over a 30-kDa Centricon Plus-70 Centrifugal Filter (Millipore) to remove excess free biotin. Biotinylated rPfCSP and S02-DsbC were fluorescently labeled through sequential addition of streptavidin conjugated to FITC (SA-FITC) or BV605 (SA-BV605), respectively, in a 4:1 molar ratio.

Wang L.T., Pereira L.S., Flores-Garcia Y., O’Connor J., Flynn B.J., Schön A., Hurlburt N.K., Dillon M., Yang A.S., Fabra-García A., Idris A.H., Mayer B.T., Gerber M.W., Gottardo R., Mason R.D., Cavett N., Ballard R.B., Kisalu N.K., Molina-Cruz A., Nelson J., Vistein R., Barillas-Mury C., Amino R., Baker D., King N.P., Sauerwein R.W., Pancera M., Cockburn I.A., Zavala F., Francica J.R, & Seder R.A. (2020). A potent anti-malarial human monoclonal antibody targets circumsporozoite protein minor repeats and neutralizes sporozoites in the liver. Immunity, 53(4), 733-744.e8.

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Isolation of Extracellular Vesicles

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Immortalized CDCs were grown to confluence at 20% O₂ at 37°C, and then cells were serum‐free at 2% O₂ at 37°C overnight after three washes. Conditioned media was collected and filtered through a 0.45 μm filter to remove apoptotic bodies and cellular debris and EVs were purified using centrifugal ultrafiltration with a 10 KDa Centricon Plus‐70 Centrifugal Filter (Millipore). Particle size and concentration were measured using NanoSight NS300 (Malvern).

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Extracellular Vesicle Isolation from Conditioned Medium

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200 mL of frozen CM was thawed in a water bath and warmed to 37°C. DNAwas digested with 100 U/mL of Benzonase® (Millipore, Burlington, United States) for 1 h, supplemented with a final MgCl₂ concentration of 2 μM at 37°C. The CM was then clarified using a 0.22 µm syringe filter (Millex-GP, Millipore, Burlington, United States) and concentrated using a Centricon® Plus-70 centrifugal filter (Millipore, Burlington, United States) with a 30 kDa cut-off. The medium was concentrated at 3,500 x g for 15–20 min and replenished until it was reduced to 10.5 mL. 10 mL of the concentrated medium was used for EV isolation and the remaining 0.5 mL were used for analysis.

Koch L.F., Best T., Wüstenhagen E., Adrian K., Rammo O, & Saul M.J. (2024). Novel insights into the isolation of extracellular vesicles by anion exchange chromatography. Frontiers in Bioengineering and Biotechnology, 11, 1298892.

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Isolation and purification of exosomes

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The culture supernatants of EO771 cells at approximately 60–70% confluence were harvested after 16 h conditioning in serum-free media (11 (link)). Exosomes were isolated as previously described (6 (link)). Briefly, cells and debris were cleared from the supernatant by centrifugation (500 g, 10 min), followed by filtration using 0.22 μm filters (Merck Millipore). Cell-free supernatants were concentrated by ultrafiltration through Centricon Plus-70 Centrifugal Filter (100 kDa; Merck Millipore), spun at 3,500 g at 4°C. Exosomes were subsequently purified by overlaying concentrated samples on qEV size exclusion chromatography columns (Izon Science Ltd.) followed by elution with PBS. Finally, the elute from qEV columns were concentrated using Amicon Ultra-4 10-kDa nominal molecular weight centrifugal filter units (Merck Millipore) to a final volume of approximately 200 µL.

Ham S., Lima L.G., Chai E.P., Muller A., Lobb R.J., Krumeich S., Wen S.W., Wiegmans A.P, & Möller A. (2018). Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling. Frontiers in Immunology, 9, 871.

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