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Centricon plus 70 centrifugal filter unit

Manufactured by Merck Group
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Sourced in United States, Ireland
About the product

The Centricon® Plus-70 Centrifugal Filter Units are a type of laboratory equipment used for the concentration and purification of macromolecules, such as proteins and nucleic acids, from complex solutions. The device utilizes a centrifugal force to facilitate the separation and concentration of the desired molecules through a selectively permeable membrane.

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Spelling variants (same manufacturer)

Centricon - 331 citations Centrifugal filter units - 318 citations Centrifugal filter - 250 citations Centricon Plus-70 - 217 citations Centricon filters - 121 citations Centricon centrifugal filter - 33 citations Centricon filter units - 18 citations Centricon units - 12 citations Filter units - 5 citations Centricon Plus-70 centrifugal - 3 citations

Similar products (other manufacturers)

Centrifugal filter unit (by Microcon) - 11 citations Centrifugal filter unit (by Centricon) - 5 citations Centrifugal filter unit (by MDMillipore) - 3 citations Centrifugal filter units (by Centriprep) - 3 citations Centricon Plus 70 (by Thermo Fisher Scientific) - 2 citations Centrifugal filter units (by Thermo Fisher Scientific) - 2 citations Centrifugal filter units (by Avantor) - 2 citations Centrifugal filter units (by Pall Corporation) - 2 citations

The spelling variants listed below correspond to different ways the product may be referred to in scientific literature.
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34 protocols using «centricon plus 70 centrifugal filter unit»

1

Ultracentrifugation of Cell Culture EVs

2024
The 10,000 × g conditioned cell culture supernatant was concentrated at 3500 × g for 30 min using Centricon® Plus‐70 Centrifugal Filter Units, MWCO 10 kDa (Merck, Cat. No.: UFC701008). The concentrated EVs mixture was used freshly or stored at −80°C for up to 2 months.
Pham C.V., Chowdhury R., Patel S., Jaysawal S.K., Hou Y., Xu H., Jia L., Zhang Y., Wang X., Duan W, & Xiang D. (2024). An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy. Journal of Extracellular Vesicles, 13(9), e12502.
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2

Phage Isolation and Purification Protocol

2024
Phages PSA04 (GenBank: MZ089728.1) and PSA34 (GenBank: MZ089739.1) were received from the University of Pittsburgh (gift from Dr. Daria Van Tyne) and phages OMKO1 (GenBank: ON631220.1) and LPS-5 (FASTA file can be shared upon request) from Yale University (gift from Dr. Paul Turner) and plated for plaque purification using the double-layer agar method as described below. Plaques were picked using a sterile pipette tip 24 h later and used to infect their respective phage hosts in 5 ml cultures. Cultures were filtered 16 h later using 0.45 and 0.22 µm syringe filters with a polyethersulfone (PES) membrane (ThermoFisher Scientific), and the flow through was tittered by plaque assay as described below. Phage lysates were stored at 4°C and used for large scale phage purifications in 1 L cultures. P. aeruginosa strains PAO1 (for phages OMKO1 and LPS-5), DVT423 (for PSA04), and DVT411 (for PSA34) were grown at mid-log phase and infected with phages at an MOI of 0.005 PFU/cell for at 37°C for 16 h with shaking at 250 rpm in LB medium supplemented with 0.001 M of CaCl2 and MgCl2. Bacteria were lysed with 1% chloroform and removed by centrifugation at 5,500 x g for 20 minutes (min). Supernatants were treated with 1 µg/ml of DNase I (Sigma) and RNase A (ThermoFisher Scientific) at 37°C for 1 h, followed by vacuum filtration using a 0.45 µm filter and 0.22 µm filter with PES membranes (ThermoFisher Scientific). Supernatants were concentrated using Centricon Plus-70 centrifugal filter units with a membrane pore size of 100,000 Da (EMD Millipore) according to the manufacturer instructions. Centrifugations were repeated until supernatants were 100-fold more concentrated compared to the initial volume.
Supernatants were loaded onto cesium chloride (CsCl) gradients (densities: 1.70, 1.50, 1.45, and 1.33 g/ml), that were prepared in SM buffer (70 (link)) and centrifuged at 90,000 x g at 4°C for 16 h. Bands were collected using a 20-gauge syringe (Fig. S1), reloaded onto a similar CsCl gradient, and centrifuged again as before to increase phage purity. CsCl was removed by extensive dialysis using dialysis buffer (50 mM NaCl, 15 mM MgCl2, 10 mM Tris pH 7.4) and dialysis Float A Lyzer devices with a pore size of 10,000 Da (Sigma). Phage identity was confirmed by PCR using phage-specific primers (Table S1). Phage particle concentration was obtained by measuring absorbance at 269 and 320 nm and calculated using the (A269 -A320) x 6 x 1016 / number of bases per virion, as described (71 (link)). Preparations were tested for endotoxins using the Limulus amoebocyte lysate test (ThermoFisher Scientific) and bacterial DNA by amplification with 16S rRNA gene primers (72 (link)). All phage preparations used in experiments had endotoxin levels below 0.2 EU/ml in 1 x 108 particles/ml.
Zamora P.F., Reidy T.G., Armbruster C.R., Sun M., Van Tyne D., Turner P.E., Koff J.L, & Bomberger J.M. (2024). Lytic bacteriophages interact with respiratory epithelial cells and induce the secretion of antiviral and proinflammatory cytokines. bioRxiv.
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3

Isolation of Small Extracellular Vesicles

2023
Per isolation procedure, 40 to 70 mL of CF urine was thawed and ultrafiltrated using 100 kDa MWCO Centricon® Plus-70 Centrifugal Filter Units (Merck Millipore Ltd., Ireland) to get rid of the solute, smaller particles and proteins. The filtrate was placed on a qEV single 70 nm original column (Izon Science Ltd., Addington, New Zealand). With these columns, 200 µL fractions were collected, starting immediately after placing the sample on the column, with filtered PBS as the elution buffer. The first mL (=dead volume) was collected starting immediately after placing 100 µL filtrate on the column. The next 500 µL was than collected containing the sEVs. The qEV columns were used according to the manufacturer’s instructions.
Jordaens S., Oeyen E., Willems H., Ameye F., De Wachter S., Pauwels P, & Mertens I. (2023). Protein Biomarker Discovery Studies on Urinary sEV Fractions Separated with UF-SEC for the First Diagnosis and Detection of Recurrence in Bladder Cancer Patients. Biomolecules, 13(6), 932.
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4

Environmental RNA Preservation and Filtration

2023
After arrival to the lab, samples were preserved by fixation with 10% volume of stop solution (5% phenol: 95% ethanol) in order to conserve environmental RNA (Feike et al., 2012 (link)) and divided into three 50 ml aliquots in 50 ml centrifugation tubes. Larger suspended solids were removed by centrifugation at 4600 g for 30 min (4 °C). With syringes, the supernatant was carefully transferred and filtered manually through 0.22 µm Sterivex™ GP Sterile Filter Units (Merck KGaA, Germany) to eliminate non-viral particles from the solution. The filtrate (3 × 50 ml) was collected into new 50 ml centrifugation tubes and concentrated using Centricon® Plus-70 Centrifugal Filter Units with a molecular weight cut-off of 10 kDa (Merck Millipore, Burlington, USA) at 3500 x g for about 30 min each step (Medema et al., 2020 (link)). Each sample was concentrated down to about <600 µl solution, which was stored at −20 C° until RNA extraction for up to 24 h.
Kisand V., Laas P., Palmik-Das K., Panksep K., Tammert H., Albreht L., Allemann H., Liepkalns L., Vooro K., Ritz C., Hauryliuk V, & Tenson T. (2023). Prediction of COVID-19 positive cases, a nation-wide SARS-CoV-2 wastewater-based epidemiology study. Water Research, 231, 119617.
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5

Exosome Isolation from Cell Culture

2023
STX-S and STX-N cells were cultured in FreeStyle medium (catalog number 12338018; Thermo Fisher) in a Multitron incubator (Infors HT). Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s laboratory-scale or large-scale purification method. For the laboratory-scale method, the supernatant was subjected to concentrating filtration using a Centricon Plus-70 centrifugal filter unit (catalog number UFC710008; Millipore) and then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (SP5; Izon). For the large-scale method, the supernatant was subjected to concentrating tangential flow filtration (TFF) on an Äkta Flux s instrument (Cytiva, USA) and then subjected to size exclusion chromatography on an Äkta Avant 25 system (Cytiva, USA).
Cacciottolo M., Nice J.B., Li Y., LeClaire M.J., Twaddle R., Mora C.L., Adachi S.Y., Chin E.R., Young M., Angeles J., Elliott K, & Sun M. (2023). Exosome-Based Multivalent Vaccine: Achieving Potent Immunization, Broadened Reactivity, and Strong T-Cell Responses with Nanograms of Proteins. Microbiology Spectrum, 11(3), e00503-23.
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Top 5 protocols citing «centricon plus 70 centrifugal filter unit»

1

Concentration and Purification of Hepatitis C Virus

Cited 1 time
Huh7.5 cells were transfected by electroporation with Jc1-derived in vitro transcripts. Culture supernatants of transfected cells were harvested 24, 48, 72 and 96 h after transfection and cleared by passing through 0.45-µm pore size filters. Supernatants were pooled and concentrated by using Centricon Plus-70 Centrifugal Filter Units (Millipore). Concentrates were loaded on top of an 80% Optiprep (Axis-Shield, Oslo, Norway) cushion in PBS and centrifuged for 4 h at 4°C and 30,000 rpm in an SW 28 Ti rotor (Beckman Coulter, Krefeld, Germany). Alternatively, supernatants were precipitated by using PEG-8,000 as described elsewhere [67] (link). Precipitate was spun down at 8,000× g for 90 min and resuspended in complete DMEM. Concentrated virus sample was collected and infectivity titer was determined by limiting dilution assay.
Romero-Brey I., Merz A., Chiramel A., Lee J.Y., Chlanda P., Haselman U., Santarella-Mellwig R., Habermann A., Hoppe S., Kallis S., Walther P., Antony C., Krijnse-Locker J, & Bartenschlager R. (2012). Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication. PLoS Pathogens, 8(12), e1003056.
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2

Isolation and Characterization of Extracellular Vesicles

Cited 1 time
In all experiments, sEVs were isolated from conditioned media and body fluids (with the exception of plasma samples in Fig. 8g) by the following procedure. Conditioned media was prepared by culturing cancer cells in media containing 2% FBS for 48 h. For each batch preparation of sEVs, a total of 360 mL of conditioned media collected from 20 × 150 mm dishes of cells at 90% confluence was used. For each purification of sEVs from biological fluid, a 1 mL sample of fluid was used. Conditioned media and biological fluids were centrifuged at 2,400 × g at 4 °C for 10 min to remove intact cells and cell debris. Thereafter, supernatants were filtered through a 0.2 μm pore size filter to exclude particles of > 200 nm in diameter and then concentrated using a Centricon® Plus-70 centrifugal filter unit with a 100 kDa nominal molecular weight limit (Millipore) to exclude soluble proteins of < 100 kDa in size. To prepare the discontinuous iodixanol gradient, solutions of iodixanol were prepared by diluting a stock solution of Optiprep™ (60% (w/v) aqueous iodixanol, Axis-Shield PoC) in buffer containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA. Concentrated conditioned media and biological fluids were mixed with 1.5 mL of Optiprep™ stock solution on the bottom of a 14 × 95 mm polyallomer ultracentrifuge tube (Beckman Coulter). The gradient was formed by stepwise layering of 3.0 mL of 40% (w/v) iodixanol solution, 2.5 mL each of 20% (w/v) and 10% (w/v) iodixanol solutions, and 2.0 mL of 5% (w/v) iodixanol solution. Centrifugation was performed at 200,000 × g at 4 °C for 18 h. Ten gradient fractions of 1.0 mL were collected from the top to bottom. The density of each fraction was determined from absorbance readings at 244 nm using a standard curve generated from serial dilutions of iodixanol solution53 (link). Individual fractions were washed with PBS, concentrated by using Centricon® filter units, and suspended in PBS for further analysis. As volumes of plasma samples of bevacizumab-treated patients were small (200 μL), sEVs were isolated from these samples (Fig. 8g) by using ExoQuick reagent (System Biosciences). Briefly, plasma samples were centrifuged at 3000 × g for 15 min to remove cells and debris. Samples (100 μL) were then diluted with the addition of 400 μL PBS and 120 μL precipitation solution, vortexed and incubated at 4 °C for 16 h. Thereafter, sEVs were precipitated by centrifugation at 1500 × g for 30 min and suspended in PBS for further analysis.
Ko S.Y., Lee W., Kenny H.A., Dang L.H., Ellis L.M., Jonasch E., Lengyel E, & Naora H. (2019). Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake. Communications Biology, 2, 386.
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3

Lentivirus Production and Transduction

Large-scale production and concentration of lentivirus were performed as previously described (Beronja et al., 2010 (link)). Briefly, 293TN cells (System Biosciences, LV900A-1) cultured in DMEM+10% FBS (Gibco) were transfected using calcium phosphate with lentiviral backbone and helper plasmids pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260) for 16 hr, then incubated in UltraCULTURE media (Lonza) for 48 hr. Supernatant was collected and concentrated using Centricon Plus-70 centrifugal filter unit (100 kDa, Millipore), then further pelleted by ultracentrifugation at 45,000 rpm for 1 hr using a MLS 50 rotor (Beckman Coulter). Viral pellet was resuspended in Viral Resuspension Buffer (VRB; 20 mM Tris pH 8.0, 250 mM NaCl, 10 mM MgCl2, 5% sorbitol) to generate lentiviral stock for intra-amniotic injections. Lentiviral transduction of primary keratinocytes in culture and of embryonic ectoderm in utero were performed as previously described with modifications (Beronja et al., 2013 (link); 2010 (link); Beronja and Fuchs, 2012 (link)). Briefly, primary keratinocytes were plated on 6-well plates and incubated in E media with 20% chelexed FBS, lentivirus, and 40 μg/ml polybrene at 37°C for 30 min, then centrifuged at 37°C and 1100xg for 30min. Centrifuged plates were rinsed with PBS and cultured in E media with 15% FBS for 2 days before processing for analyses. For in utero lentiviral transduction of embryonic ectoderm, pregnant mice were imaged one day before surgery using a Vevo 1100 animal ultrasound imager with a MS550D transducer (Visualsonics) to precisely stage the embryos. This ensured that lentiviral transduction was performed on E9.25–9.75 day embryos. Under ultrasound guidance, 1uL of lentivirus was injected into the amniotic cavity on the ventral side of each embryo with a Nanoject II microinjector (Drummond).
Cai E.Y., Kufeld M.N., Schuster S., Arora S., Larkin M., Germanos A.A., Hsieh A.C, & Beronja S. (2020). Selective translation of cell fate regulators mediates tolerance to broad oncogenic stress. Cell stem cell, 27(2), 270-283.e7.
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4

Extracellular Vesicle Isolation Protocol

The purified conditioned cell media of six T75 flasks and blank controls (90 mL) were concentrated to a volume of maximum 500 µL using a 100 kDa nominal molecular weight limit Centricon Plus-70 Centrifugal Filter Unit (Sigma-Aldrich, Saint Louis, MO, USA, #UFC710008) according to the manufacturer’s protocol. The concentrated conditioned cell medium or 500 µL of PPP were loaded onto a qEVoriginal 70 nm column (Izon Science Ltd., Lyon, France, #SP1), and immediately fractions of 500 µL were collected with PBS as elution buffer. Fractions 1 to 4 after the void volume were pooled (2 mL). Cell-line derived fractions were transferred to a 5 mL open-top thinwall polyallomer tube and centrifuged at 4 °C; 100,000 rcf for 65 min using an Optima XPN-80 ultracentrifuge equipped with a swinging bucket SW55 Ti rotor. The pellet was resuspended in 500 µL of PBS. PPP-derived fractions were immediately used for DNA extraction (see Section 4.8).
Van Hoof R., Deville S., Hollanders K., Berckmans P., Wagner P., Hooyberghs J, & Nelissen I. (2022). Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity. International Journal of Molecular Sciences, 23(24), 16052.
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5

Exosome Isolation from Cell Culture

STX-S and STX-N cells were cultured in FreeStyle medium (catalog number 12338018; Thermo Fisher) in a Multitron incubator (Infors HT). Subsequently, cells and cell debris were removed by centrifugation, while microvesicles and other extracellular vesicles larger than ~220 nm were removed by vacuum filtration. Next, exosomes were isolated using either Capricor’s laboratory-scale or large-scale purification method. For the laboratory-scale method, the supernatant was subjected to concentrating filtration using a Centricon Plus-70 centrifugal filter unit (catalog number UFC710008; Millipore) and then subjected to size exclusion chromatography (SEC) using a qEV original SEC column (SP5; Izon). For the large-scale method, the supernatant was subjected to concentrating tangential flow filtration (TFF) on an Äkta Flux s instrument (Cytiva, USA) and then subjected to size exclusion chromatography on an Äkta Avant 25 system (Cytiva, USA).
Cacciottolo M., Nice J.B., Li Y., LeClaire M.J., Twaddle R., Mora C.L., Adachi S.Y., Chin E.R., Young M., Angeles J., Elliott K, & Sun M. (2023). Exosome-Based Multivalent Vaccine: Achieving Potent Immunization, Broadened Reactivity, and Strong T-Cell Responses with Nanograms of Proteins. Microbiology Spectrum, 11(3), e00503-23.
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