0.45 um filter

Manufactured by Merck Group

The 0.45 um filter is a laboratory equipment used for filtration purposes. It is designed to remove particles and contaminants from liquids or gases that are 0.45 micrometers or larger in size. The filter is often made of materials such as cellulose, nylon, or polyethersulfone, and is commonly used in various industries, including pharmaceuticals, biotechnology, and water treatment, to purify and clarify samples.

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Lab products found in correlation

Polybrene, by Merck Group (2 mentions) Polybrene, by Solarbio (1 mentions) Puromycin, by Solarbio (1 mentions) Centrifugal filter unit, by Merck Group (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Geneticin g418, by Thermo Fisher Scientific (1 mentions) Fugene 6, by Promega (1 mentions) Pvsv g, by Addgene (1 mentions) Macrosep advance centrifugal device, by Pall Corporation (1 mentions) Pspax2, by Addgene (1 mentions) Quicktiter lentivirus titer kit, by Cell Biolabs (1 mentions)

6 protocols using 0.45 um filter

Lentiviral Transduction for Gene Modulation

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Lentiviruses for gene knockdown or overexpression experiments were produced by transient co-transfection of 293T cells for 48 h with PLKO.1, pREV, pGag and pVSVG (2:2:2:1 ratio) or pCDH, pspax2 and pmd2.g (2:2:1 ratio). Lentiviral supernatants were filtered by 0.45um filter (Millipore) and supplemented with 8 µg/ml polybrene (Solarbio) before incubation with the target cells, followed by selection with 10 µg/ml puromycin (Solarbio). The targeting sequences used are shown in Additional file 6: Table S4.

Gao E., Sun X., Thorne R.F., Zhang X.D., Li J., Shao F., Ma J, & Wu M. (2023). NIPSNAP1 directs dual mechanisms to restrain senescence in cancer cells. Journal of Translational Medicine, 21, 401.

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Establishment of Inducible Knockdown

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All vectors were derived from the Murine Stem Cell Virus (MSCV, Clontech) retroviral vector backbone. miRE-based shRNAs were designed and cloned as previously described (26 (link)) into the LT3GEPIR (TRE3G-GFP-miRE-PGK-PuroR-IRES-rtTA3) vector (80 (link)). The mouse Foxh1 cDNA was cloned in a pVXL-hygromycin-mFoxh1 vector (provided by Yilong Zou, Massagué lab, MSKCC). All constructs were verified by sequencing. Lentiviruses were produced by co-transfection of 293T cells with 10 ug LT3GEPIR construct and helper vectors (3.75 ug psPAX2 and 1.25 ugVSV-G). For retroviral infection PlatinumE cells were plated in 15 cm diameter dishes each transfected with 54 ug of MSCV vectors and 2.7ug of VSV-G and 8.1ug of pCl-eco. Transfection of packaging cells was performed using HBS and CaCl₂ or Mirus transfection reagent (MIR 2304). Viral supernatants were passed through a 0.45 um filter (Millipore) and concentrated with Centrifugal Filter units (Millipore) to obtain high virus titer. Concentrated virus was supplemented with 8 ug/ml of polybrene (Sigma) before adding to target cells.

Loizou E., Banito A., Livshits G., Ho Y.J., Koche R.P., Sánchez-Rivera F.J., Mayle A., Chen C.C., Kinalis S., Bagger F.O., Kastenhuber E.R., Durham B.H, & Lowe S.W. (2019). A gain-of-function p53 mutant oncogene promotes cell fate plasticity and myeloid leukemia through the pluripotency factor Foxh1. Cancer discovery, 9(7), 962-979.

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Retroviral Vectors for Expressing shRNA and cDNA

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All vectors were derived from the Murine Stem Cell Virus (MSCV, Clontech) retroviral vector backbone. miRE-based shRNAs were designed and cloned as previously described (Zuber et al., 2011a (link)) into the LT3GEPIR (TRE3G-GFP-miRE-PGK-PuroR-IRES-rtTA3) vector (Fellmann et al., 2013 (link)) (Table S6). The mouse KDM2B cDNA was subcloned from pVXL-Tomato-KDM2B vector (provided by A. Tzatsos) into MSCV-Neo using an EcoRI site. The JmjC and ZF-CxxC were generated from the wild-type KDM2B vector by site directed mutagenesis (Q5 site-directed mutagenesis kit, New England Biolabs). The short isoform of KDM2B was amplified by PRC from M5SS1 cDNA and cloned into MSCV-hygro. All constructs were verified by sequencing. For CRISPR editing constructs see CRISPR/Cas9 genome editing section. Lentiviruses were produced by co-transfection of 293T cells with 10 ug LT3GEPIR construct and helper vectors (6.5ug psPAX2 and 2.5ugVSV-G). For retroviral infection 293T-gag-pol cells were transduced with 20 ug of MSCV vectors and 2.5ug of VSV-G. Transfection of packaging cells was performed using Polyethylenimine (PEI) (Polysciences, 23966-2) by mixing with DNA in a 3:1 ratio. Viral supernatants were collected 48 after transfection, filtered thorough a 0.45 um filter (Millipore) and supplemented with 4 ug/ml of polybrene (Sigma) before adding to target cells.

Banito A., Li X., Laporte A.N., Roe J.S., Sanchez-Vega F., Huang C.H., Dancsok A.R., Hatzi K., Chen C.C., Tschaharganeh D.F., Chandwani R., Tasdemir N., Jones K.B., Capecchi M.R., Vakoc C.R., Schultz N., Ladanyi M., Nielsen T.O, & Lowe S.W. (2018). The SS18-SSX oncoprotein hijacks KDM2B-PRC1.1 to drive synovial sarcoma. Cancer cell, 33(3), 527-541.e8.

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Generation of Stable cDNA Overexpression Lines

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To generate stable cDNA overexpression lines, cDNAs bearing the indicated mutations and the wild-type counterparts were synthesized by Genewiz except where noted. For TBK1 (NCBI), all constructs included an N-terminal FLAG epitope tag and contained wobble mutations to render the cDNA insensitive to the TBK1-g1 sgRNA. For TAX1BP1 (NCBI), all constructs included an N-terminal MYC epitope and the cDNA sequence was codon optimized. Constructs were then subcloned into the pXP1510 lentiviral vector, allowing for cDNA expression driven by the EF1-alpha promoter. To generate stable cell lines, 8 × 10⁵ 293T cells were plated in 6-well plates. The next day, cells were transfected with lentiviral packaging mix (1 ug ΔVPR (Δ8.9) and 0.25 ug VSV-G) along with 1 ug of the pXP1510 vector using FuGene 6 (Promega). Supernatant was removed from 293T cells after 48 hr, centrifuged at 2000 rpm for 5 min and then syringe filtered using a 0.45 um filter (Millipore). Polybrene was then added to a final concentration of 8 ug/ml and target cells were infected overnight. Cells were then allowed to recover for 24 hr in DMEM/10% FBS before being selected with 1 mg/ml neomycin (G418:Geneticin, Life Technologies) for 72 hr.

Goodwin J.M., Dowdle W.E., DeJesus R., Wang Z., Bergman P., Kobylarz M., Lindeman A., Xavier R., McAllister G., Nyfeler B., Hoffman G, & Murphy L.O. (2017). Autophagy-independent lysosomal targeting regulated by ULK1/2-FIP200 and ATG9. Cell reports, 20(10), 2341-2356.

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Lentiviral Construct Production and Cell Line Generation

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The recombinant lentiviral constructs LentiCRISPRv2, as well as assistant vectors: pVSVg (AddGene 8454) and psPAX2 (AddGene 12,260) were co-transiently transfected into HEK293T cells. Viral supernatants were collected 48 h later, centrifuged at 500 g for 5 min to remove debris, clarified by filtrated through 0.45um filter (Millipore), and then concentrated with Macrosep® Advance Centrifugal Device (100 K, PALL Corporation). Lentivirus titers were determined with the QuickTiter lentivirus titer kit (Cell Biolabs, San Diego, CA). The concentrated virus was used to infect Huh7 and SK-HEP-1 cells at an MOI of 50 with 4 μg/ml polybrene. Infected Huh7 and SK-HEP-1 cells were then subjected to puromycin selection for the establishment of stable cell lines.

Xie X., Wang X., Liao W., Fei R., Wu N., Cong X., Chen Q., Wei L., Wang Y, & Chen H. (2018). MYL6B, a myosin light chain, promotes MDM2-mediated p53 degradation and drives HCC development. Journal of Experimental & Clinical Cancer Research : CR, 37, 28.

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Pseudotyping HIV-1 with VSVg for Infectivity

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To generate HIV-1 particles pseudotyped with VSVg, 293T cells seeded in a 10-cm dish at ~70% confluency was transfected with either 7ug of R7∆Env-GFP or NL4.3Luc-mCherry and 3ug of pCMV-VSVg using polyethylenimine (PEI). Media was replaced 12 hours after transfection. Viruses were harvested 48 hours after transfection and filtered through a 0.45-um filter (Milipore). To measure infectivity, equal amounts of NL4.3Luc-mCh or R7-GFP virus, as measured by RT activity, was added to cells and a synchronized infection was performed by spinoculation at 13°C for 2 hours at 1,200 x g. After spinoculation, virus-containing media was removed and replaced with 37°C media. Infectivity was measured 48 hours after infection using either luciferase assay to measure presence of firefly luciferase or flow cytometry to measure the number of GFP-positive cells.

Badieyan S., Lichon D., Andreas M.P., Gillies J.P., Peng W., Shi J., DeSantis M.E., Aiken C.R., Böcking T., Giessen T.W., Campbell E.M, & Cianfrocco M.A. (2023). HIV-1 binds dynein directly to hijack microtubule transport machinery. bioRxiv.

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