Amicon ultra 15 100k

Manufactured by Merck Group

Sourced in Germany, United States

The Amicon Ultra-15 100K is a centrifugal filter device used for sample concentration and buffer exchange. It features a molecular weight cutoff of 100 kDa, which allows the retention of molecules above this size while allowing smaller molecules to pass through.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Benzonase, by Merck Group (1 mentions) Pei max, by Polysciences (1 mentions) Prrlsin cppt pgk, by Addgene (1 mentions) Ufc910024, by Merck Group (1 mentions) 0.22 μm pvdf membrane, by Merck Group (1 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions) Sw60ti rotor, by Beckman Coulter (1 mentions)

Close spelling variants of this product

Amicon Ultra-15 Centrifugal Filter Device 100K Amicon Ultra-15 100K filters Amicon Ultra-15 100K filter units 100K Amicon® Ultra 15 Centrifugal Filter Units Amicon Ultra-15 100K columns Amicon Ultra-15 Ultracel-100K Amicon Ultra 100K Amicon Ultra-15 Amicon Ultra

15 protocols using amicon ultra 15 100k

Manipulating Neuronal Stem Cell Differentiation

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LV-CLU shRNA transfection was performed in order to evaluate the effect of CLU on the development and differentiation of NSEs exposed to acoustic stimuli. For the lentiviral-mediated CLU shRNA delivery, shRNAs were designed, chemically synthesized and PAGE-purified, free of RNase contamination, according to the instructions of the manufacturer (GeneChem, Shanghai, China). They were then ligated into the pSuppres-sorNeo plasmid (provided by Dr J.S. Yan, Department of Microbiology, the Fourth Military Medical University) as previously described. Scramble oligonucleotides were used as a negative control.
Transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions as follows: 20 μg of lentiviral vector carrying shRNA and 100 μl Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were mixed and incubated with HEK293T cells (obtained from the Chinese Academy of Medeical Science, Beijing, China) at 37°C, 5% CO₂ for 48 h. Cell supernatants were collected and concentrated using a 0.45 μm filter (Amicon Ultra-15 100K; Millipore, Billerica, MA, USA); the recombinant virus was stored at −80°C until use. Newborn rats were administered an intraperitoneal injection of 2.5 μg pSuppressorNeo in 1 ml of DMEM. The treated rats were exposed to environmental noise as described above. The effects of transfection were confirmed by RT-qPCR and western blot analysis.

XUE T., WEI L., ZHA D.J., QIAO L., LU L.J., CHEN F.Q, & QIU J.H. (2015). Exposure to acoustic stimuli promotes the development and differentiation of neural stem cells from the cochlear nuclei through the clusterin pathway. International Journal of Molecular Medicine, 35(3), 637-644.

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Exosomes Isolation from Cell Culture

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Exosomes were isolated from the supernatants of cultured cells with an SBI ExoQuick‐TC Kit (System Biosciences, Mountain View, CA, USA) according to the manufacturer's protocol with minor modifications. The supernatants were purified in advance using Amicon Ultra15 100K ultrafilter devices (Millipore, Billerica, MA, USA) to remove nonexosomal proteins. For proteomic analysis and electron microscopy, the initial pellet was resuspended in sterile PBS. The resuspended pellet was isolated again with an ExoQuick‐Tc Kit using the same procedure to further remove nonexosomal proteins.

Jin H., Liu P., Wu Y., Meng X., Wu M., Han J, & Tan X. (2018). Exosomal zinc transporter ZIP4 promotes cancer growth and is a novel diagnostic biomarker for pancreatic cancer. Cancer Science, 109(9), 2946-2956.

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HBV Infection of Liver Organoids

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The supernatant produced by infected organoids at 4–8 days post infection was collected and concentrated using Amicon Ultra-15 100K (Milipore). Human liver organoids were resuspended using either concentrated HBV virus or HI control. After an hour of spinoculation at 32°C, 600 xg, the plate was incubated at 37°C overnight and then washed and seeded in BME following the HBV infection and culturing protocol.

De Crignis E., Hossain T., Romal S., Carofiglio F., Moulos P., Khalid M.M., Rao S., Bazrafshan A., Verstegen M.M., Pourfarzad F., Koutsothanassis C., Gehart H., Kan T.W., Palstra R.J., Boucher C., IJzermans J.N., Huch M., Boj S.F., Vries R., Clevers H., van der Laan L.J., Hatzis P, & Mahmoudi T. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. eLife, 10, e60747.

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Production of Recombinant HEV-Like Particles

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To produce the wild-type and chimeric HEV-LP, Tn5 cells were infected with recombinant baculoviruses at a multiplicity of infection (moi) of 5. The culture media were harvested at 7 days post-infection. The intact cells, cell debris and progeny baculoviruses were removed by centrifugation at 10,000 × g for 40 min. The supernatants were further centrifuged at 100,000 × g for 3 h. The resulting pellets suspended in phosphate buffered saline (PBS) overnight were centrifuged on a 10–40% sucrose density gradient at 38,000 × g for 2 h. The HEV-LP were recovered from middle fractions of the sucrose density gradient. Finally, the collected HEV-LP were dialyzed and concentrated by ultrafiltration using an Amicon Ultra-15 100K (Merck Millipore, Billerica, MA).

Shima R., Li T.C., Sendai Y., Kataoka C., Mori Y., Abe T., Takeda N., Okamoto T, & Matsuura Y. (2016). Production of hepatitis E virus-like particles presenting multiple foreign epitopes by co-infection of recombinant baculoviruses. Scientific Reports, 6, 21638.

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Recombinant AAV8 Production and Administration

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To produce rAAV8, a triple cotransfection procedure was used to introduce an rAAV vector plasmid (pAAV-CMV-mHNF4α or pAAV-CMV-GFP) together with pXR8, AAV8 helper plasmid carrying AAV rep and cap genes and pXX6-80, and Ad helper plasmid at a 1:1:1 molar ratio (51 (link)).
Briefly, HEK293T cells were transfected using poly-ethylenimine (PEI; linear; molecular weight, 25,000; Polysciences, Inc.), and the medium was replaced at 18 hours after transfection. Cells were harvested 72 hours after transfection, subjected to 3 rounds of freeze–thawing, and then digested with 100 U/mL Benzonase (EMD Millipore) at 37^°C for 1 hour. Viral vectors were purified by iodixanol (Serumwerk Bernburg AG) gradient ultracentrifugation (52 (link)), followed by further concentration using Amicon ultra-15 100K (100,000-molecular-weight cutoff, Merck Millipore) and washed with PBS (−/−). The final concentration of rAAV8 particles was 2.78E+10 vg per microliter (AAV-CMV-mHNF4α) and 2.35E+10 vg per microliter (pAAV- CMV-GFP). Mice were injected via tail vein with 5E11 vg 48 hours following inoculation with cancer cells.

Goldman O., Adler L.N., Hajaj E., Croese T., Darzi N., Galai S., Tishler H., Ariav Y., Lavie D., Fellus-Alyagor L., Oren R., Kuznetsov Y., David E., Jaschek R., Stossel C., Singer O., Malitsky S., Barak R., Seger R., Erez N., Amit I., Tanay A., Saada A., Golan T., Rubinek T., Sang Lee J., Ben-Shachar S., Wolf I, & Erez A. (2023). Early Infiltration of Innate Immune Cells to the Liver Depletes HNF4α and Promotes Extrahepatic Carcinogenesis. Cancer Discovery, 13(7), 1616-1635.

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Exosome Isolation from Cell Cultures

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After cell cultures reached 80% confluent, A549 cells or c-Jun-KO A549 cells were washed with PBS and incubated with FBS free RPMI 1640 medium for 48 h, or were treated with SP600125 for 48 h in FBS free medium. Exosomes were isolated from the medium by Total Exosome Isolation Regent as described in the manufacture’s manual. Briefly, the harvested supernatants were filtered through 0.22 μm membrane to remove cells and debris, then concentrated using Amicon Ultra-15 100K centrifuge tube (MERCK Millipore). After transferring the cell-free culture media to a new tube, 0.5 volumes of the Total Exosome Isolation Reagent were added. After incubation at 4°C overnight, the suspension was centrifuged at 10,000×g for 1 h to remove the supernatant. The resulting exosomes were re-suspended in PBS and stored at 4°C to be available for use.

Shao C., Huang Y., Fu B., Pan S., Zhao X., Zhang N., Wang W., Zhang Z., Qiu Y., Wang R., Jin M, & Kong D. (2021). Targeting c-Jun in A549 Cancer Cells Exhibits Antiangiogenic Activity In Vitro and In Vivo Through Exosome/miRNA-494-3p/PTEN Signal Pathway. Frontiers in Oncology, 11, 663183.

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Production and Concentration of HIV-1 Pseudoviral Particles

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To obtain HIV-1 pseudoviral particles, HEK293FT cells were seeded in a 75 cm² culture flask and cotransfected with a pSG3ΔENV vector (6.9 μg) and a plasmid encoding the corresponding Env protein from the global HIV-1 panel (3.5 μg) [11 (link)]. Panel of Global HIV-1 Env Clones (cat# 12670) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from Dr. David Montefiori. PEI MAX (Polysciences Inc., Warrington, PA, USA) was used for transfection. The DNA:PEI ratio was 1:3. The procedure for the transfection and collection of the cell supernatant containing pseudoviral particles is described in [12 (link)]. Fifteen milliliters of viral supernatant were added onto Amicon Ultra-15 (100K) (Merck Millipore, Darmstadt, Germany) and passed through the filter by centrifugation until the residual volume reached 1.5 mL. Then, diafiltration with 10 mL of 1× dPBS per 1 Amicon filter was performed. Finally, the solution was spun to a 1.5 mL volume. The concentrated pseudoviral particles were stored in small aliquots at −70 °C.

Kochina E.A., Urusov F.A., Kruglov A.A., Glazkova D.V., Shipulin G.A, & Bogoslovskaya E.V. (2022). Double and Triple Combinations of Broadly Neutralizing Antibodies Provide Efficient Neutralization of All HIV-1 Strains from the Global Panel. Viruses, 14(9), 1910.

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Conjugated Polymer Nanoparticle Preparation

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Solid-state thin films were prepared from 5 mg/ml solutions, using a spin speed of 1,200 rpm. Conjugated polymer nanoparticles were prepared by the solvent displacement technique, with a weight ratio of 1:10 (conjugated polymer:PEG_5K-PLGA_55K) (Abelha et al., 2019 (link)). A total of 4 ml of THF solution (0.9 mg/ml polymers) was added dropwise to 20 ml of water at room temperature, and the mixture was stirred until THF had completely evaporated. Formulations containing 100% PEG_5K-PLGA_55K and 100% CN-PPV were also prepared with the same polymer concentration. The resulting solutions had a total solid content of 0.2 mg/ml, which were concentrated to a minimum of 1.0 mg/ml using Amicon®Ultra-15 100K centrifugal filter devices (Merck Millipore, Tullagreen, Ireland).

Feyen P.L., Matarèse B.F., Urbano L., Abelha T.F., Rahmoune H., Green M., Dailey L.A., de Mello J.C, & Benfenati F. (2022). Photosensitized and Photothermal Stimulation of Cellular Membranes by Organic Thin Films and Nanoparticles. Frontiers in Bioengineering and Biotechnology, 10, 932877.

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Lentiviral Transduction of PR Variants

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Coding sequences for PR_A, PR_Ai, PR_A2mut, PR_Ai2mut, PR_A3mut, and PR_Ai3mut were recloned into the lentiviral vector pRRLSIN.cPPT.PGK (Addgene plasmid #12252; a gift from Dr Trono) generating pLVPR_A, pLVPR_Ai, pLVPR_A2mut, pLVPR_Ai2mut, pLVPR_A3mut, and pLVPR_Ai3mut, respectively. Lentiviral particles were produced by the transient transfection of HEK293T cells by Eurogen (Moscow, Russia) as described elsewhere [42 (link)] and concentrated 10-fold with Amicon Ultra-15 100K centrifuge concentrators (Merck-Millipore, Darmstadt, Germany). Infectious viral particles were formed only by pLVPR_A3i; their titer was determined on HT1080 cells with quantitative real-time PCR [42 (link)] using standard samples of HT-1080 DNA with a known number of viral genome copies.

Petkov S., Kilpeläinen A., Bayurova E., Latanova A., Mezale D., Fridrihsone I., Starodubova E., Jansons J., Dudorova A., Gordeychuk I., Wahren B, & Isaguliants M. (2022). HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells. Cancers, 15(1), 238.

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High-Titer SARS-CoV-2 Virus Production

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SARS-CoV-2 (BetaCoV/Australia/VIC01/2020 (Passage 2)) was obtained from the Victorian Infectious Diseases Reference Laboratory at the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia [20 (link)]. A Master stock (Passage 3) and Working stocks (Passage 4) were propagated in Vero/hSLAM cell line. To generate high titre stocks for aerosol challenge, the virus was first concentrated using Amicon^® Ultra-15 100 k centrifugal filter units for 25 min at 4000× g, at 4 °C (UFC910024, Merck Millipore, Watford, UK) prior to a final purification step through a 30% (w/v) sucrose cushion at 179,200× g for 2 h, at 4 °C. Genome stability was verified following the passage using an amplicon-based Illumina whole genome sequencing approach with the Liverpool protocol/primers [21 (link)]. Sub-consensus variant analysis was conducted using LoFreq [22 (link)]. Virus was enumerated using plaque assay or Reed & Muench TCID₅₀ method [23 (link)] in Vero C1008 cells, as indicated. The presence of replicating virus was assessed in the lungs, liver, spleen, kidney, brain, and blood by plaque assay and a further blind passage in Vero C1008 cells for 7 days.

Ireland R.E., Davies C.D., Keyser E., Findlay J.S., Eastaugh L., Laws T.R., Salguero F.J., Hunter L, & Nelson M. (2022). Histopathological and Immunological Findings in the Common Marmoset Following Exposure to Aerosolized SARS-CoV-2. Viruses, 14(7), 1580.

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