0.45 μm filtration

Manufactured by Merck Group

Sourced in United States

The 0.45 μm filtration product is a laboratory equipment used for the filtration of liquid samples. It is designed to remove particles, microorganisms, and other contaminants larger than 0.45 micrometers in size from the sample. This product serves as a crucial tool in various scientific and industrial applications that require a high level of sample purity and clarity.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions) Pspax2, by Addgene (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) High glucose dmem, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Chloroquine, by Merck Group (1 mentions) Forskolin, by Tokyo Chemical Industry (1 mentions) Pei max, by Polysciences (1 mentions) Himac cr21n, by Hitachi (1 mentions) Lenti x solution, by Takara Bio (1 mentions) Plenti puro vector, by Addgene (1 mentions) Pcdna3.1 spike, by Addgene (1 mentions) Pcdna3.1 vsv g, by Addgene (1 mentions) Pcdna3.1 empty, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Polybrene, by Merck Group (1 mentions) C18 column, by Agilent Technologies (1 mentions) 1525 binary hplc pump, by Waters Corporation (1 mentions) C18 column, by Merck Group (1 mentions)

Close spelling variants of this product

Millipore filtration MillexHV unit 0.45 μm (2 citations)

10 protocols using 0.45 μm filtration

HEK293T Lentiviral Production Protocol

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HEK293T cells (CRL-11268, ATCC) were maintained at 37°C and 5% CO₂ in a humidified atmosphere incubator with a culture media containing DMEM-high glucose (Gibco), 10% fetal bovine serum (ThermoFisher), 1% penicillin-streptomycin (10,000 U/ml, Gibco), and 1% sodium pyruvate (100 mM, Gibco). Twenty-four hours before transfection, HEK293T cells were seeded at a density of 1.9 × 10⁷ per T182 flask.
Lentiviruses were produced by transfecting the HEK293T cells with the shKIF3B and shScrambled vectors expressing GFP and three helper plasmids (pVSVg, pRRE, and pREV; Dull et al., 1998 (link)). HEK293T cells were transfected with Lipofectamine 2000 at a ratio of pshKIF3B:pRRE:pREV:pVSVg = 20:13:7:5 (ThermoFisher), following the manufacturer’s instructions. The virus-laden medium was harvested 48 h after transfection and subsequently filtered with a 1,000-rpm centrifuge and a 0.45 μm filtration (Millipore). The filtered medium was then overlaid on a 10% sucrose-PBS cushion and spun at 25,000-rpm at 4°C for 2¹/₂ h. After centrifugation, the supernatant was carefully decanted, and the tube was placed on Kimwipes for 5 min to drain. Cold PBS was added to the centrifuge tube and then the tube was placed at 4°C overnight with a parafilm cover for viral recovery.

Joseph N.F., Grinman E., Swarnkar S, & Puthanveettil S.V. (2020). Molecular Motor KIF3B Acts as a Key Regulator of Dendritic Architecture in Cortical Neurons. Frontiers in Cellular Neuroscience, 14, 521199.

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CAR T-cell Development with scFv Targeting

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CAR constructs have been developed with an scFv for either TAG-72³⁷ or CD47³⁷ ⁴² (link) as described in Boyd et al.³⁷ Following a conventional human secretion signal leader, both scFvs were constructed in a VH-linker-VL orientation with a 15-residue (Gly4Ser ×3) linker. The CAR constructs used either human CD8 or CD28 as hinge or TM regions, and used CD28, 4-1BB, and CD3ζ cytoplasmic signaling domains. The P2A sequence, a signal sequence directing proteolytic cleavage, was used to direct bicistronic expression of EGFP or the CD47 CAR. The second-generation lentiviral packaging system was used to produce the lentiviral vectors for CAR transduction. Briefly, 293T cells were plated onto poly-l-lysine-coated tissue culture plates (Sigma-Aldrich, St. Louis, MO, USA). The lentiviral transfer vector DNA, together with packaging and envelope plasmid DNA, was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Viral supernatant was collected after 48 h and cleared by centrifugation followed by 0.45-μm filtration (Millipore, Burlington, MA, USA). Concentration of lentivirus using ultracentrifugation was performed with a SORVALL Discovery 100 SE centrifuge (90 min at 20,000 × g). Virus pellets were resuspended in PBS and stored at −80°C until use.

Shu R., Evtimov V.J., Hammett M.V., Nguyen N.Y., Zhuang J., Hudson P.J., Howard M.C., Pupovac A., Trounson A.O, & Boyd R.L. (2021). Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer. Molecular Therapy Oncolytics, 20, 325-341.

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Lentivirus Production and Purification

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Lentiviruses were produced by transfecting the HEK293T cells with the pFUGW vectors expressing GFP and three helper plasmids (pVSVg, RRE, and REV)14 (link). The transfections were carried out using the Polyethylenimine (PEI) method with the ratio at PEI:pFUGW:pVSVg:RRE:REV = 24:3:1:2:2. The virus-containing medium was harvested 48 or 72 hours after transfection and subsequently pre-cleaned with a 3,000 g centrifuge and a 0.45 μm filtration (Millipore). The virus-containing medium was overlaid on a sucrose-containing buffer (50 mM Tris-HCl, pH 7.4,100 mM NaCl, 0.5 mM ethylene diamine tetra acetic acid [EDTA]) at a 4:1 v/v ratio and centrifuged at the indicated RCF at 4 °C. After centrifugation, the supernatant was carefully removed, and the tube was placed on the tissue paper for 3 minutes. Phosphate Buffered Saline (PBS) was added to the semi-dried tube for re-suspension, and then the tube was placed in the 4 °C fridge with a cover for recovery overnight.

Jiang W., Hua R., Wei M., Li C., Qiu Z., Yang X, & Zhang C. (2015). An optimized method for high-titer lentivirus preparations without ultracentrifugation. Scientific Reports, 5, 13875.

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Lentiviral Vector Production Protocol

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Recombinant lentivirus was produced as previously described³⁴ (link) with some modification. Briefly, 293FT cells were cultured on a collagen-coated 10-cm dish (Iwaki, Shizuoka, Japan) with 80%–90% confluency. The culture medium was replaced to D10 containing 25 μM chloroquine (Sigma, Darmstadt, Germany) without antibiotics. Regarding vectors packed with ΔNRF, the following plasmid amounts were used: 15 μg lentiviral vector plasmid, 10 μg ΔNRF, and 5 μg pMD.G. The plasmids were co-transfected into 293FT cells with polyethyleneimine (PEI) MAX (Polysciences, Warrington, PA) at a DNA-to-PEI ratio of 2:1. The supernatant was replaced with D10 containing 5 mM sodium butylate (Wako Pure Chemicals Industries, Osaka, Japan) and 10 μM forskolin (Tokyo Chemical Industry, Tokyo, Japan). The culture supernatants containing recombinant lentiviruses were harvested at 48 h after transfection and cleared by centrifugation and 0.45-μm filtration (Millipore, Burlington, MA). The recombinant lentiviruses were concentrated by high-speed centrifugation at 18,000 rpm for 3 h using Himac CR21N (Hitachi Koki, Tokyo, Japan). The lentiviral titers were measured on HeLa cells based on the percentage of GFP-positive cells by flow cytometry.

Nunoya J.I., Masuda M., Ye C, & Su L. (2019). Chimeric Antigen Receptor T Cell Bearing Herpes Virus Entry Mediator Co-stimulatory Signal Domain Exhibits High Functional Potency. Molecular Therapy Oncolytics, 14, 27-37.

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Production of SARS-CoV-2 Pseudotyped Lentiviruses

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SARS CoV-2 pseudotyped lentiviruses were produced by transfecting the 293T cells with the pLenti-Puro vectors (Addgene) expressing Luciferase or β-Galactosidase, with pcDNa3.1 vector expressing SARS-CoV-2 spike (BEI repository) and the helper plasmid pSPAX2 (Addgene). The VSV-G and empty lentiviruses were produced by replacing pCDNA3.1-Spike with pcDNA3.1-VSV-G or pCDNA3.1 empty vector, respectively (Addgene). The transfections were carried out using the Polyethylenimine (PEI) method with the ratio at PEI:pLenti:pcNDA3.1-Spike:pSPAX2 = 14:2:2:1 or PEI:pLenti:pVSV-G/pcNDA3.1:pSPAX2 = 10:1:0.5:3. The virus-containing medium was harvested 72 hours after transfection and subsequently pre-cleaned by centrifugation (3,000 g) and a 0.45 μm filtration (Millipore). The virus-containing medium was concentrated by using a LentiX solution (TakaraBio) a 10:1 v/v ratio and centrifuged at the indicated RCF at 4 °C. After centrifugation, the supernatant was carefully removed and the tube was drained on the tissue paper for 3 minutes. Dulbecco’s modified Eagles medium containing 4.5 g/l Glucose (DMEM) was added to the semi-dried tube for re-suspension and then stored at −80 °C.

Scaglione A., Opp S., Hurtado A., Lin Z., Pampeno C., Noval M.G., Thannickal S.A., Stapleford K.A, & Meruelo D. (2021). Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity.. bioRxiv.

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Lentiviral Transduction of Neurons

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Lentiviral expression vectors and three helper plasmids (pRSV-REV, pMDLg/pRRE and pVSVG) were co-transfected into HEK293 cells. The transfections were carried out using the polyethylenimine (PEI, 1 mg/ml in ddH₂O) method with the ratio at PEI:pFUGW:pVSVg:RRE:REV = 24:3:1:2:2. The virus-containing medium was harvested 48 h after transfection and subsequently cleaned with a 3000 g centrifuge and a 0.45 μm filtration (Millipore). The virus was then concentrated by a sucrose-containing buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5 mM ethylene diaminetetraacetic acid [EDTA]) at a 4:1 v/v ratio and centrifuged at 4°C. For re-suspension the virus, Phosphate Buffered Saline (PBS) was added to the tube at the fridge with a cover for recovery overnight. All steps were performed under level II biosafety conditions. Neurons were infected with lentiviruses at days in vitro (DIV) 5–6 and analyzed at DIV 13–14.

Yu Y., Chen S., Mo X., Gong J., Li C, & Yang X. (2018). Accessory and Central α-helices of Complexin Selectively Activate Ca2+ Triggering of Synaptic Exocytosis. Frontiers in Molecular Neuroscience, 11, 61.

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Lentiviral Transduction of HEK293T Cells

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HEK293T cells were thawed from liquid nitrogen and cultured in DMEM containing 10% FBS, growing and passage at least three times. Lentiviruses were produced by transfecting the HEK293T cells with knocking down plasmid shFN1 or shATF3, and the psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259). The transfections were carried out using the Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The virus-containing medium was harvested 48 or 72 h after transfection and subsequently pre-cleaned with a 3,000×g centrifuge and a 0.45 μm filtration (Millipore). The viruses were used for titration and infection freshly or stored at -80 °C freezer.

Wang H., Guo S., Kim S.J., Shao F., Ho J.W., Wong K.U., Miao Z., Hao D., Zhao M., Xu J., Zeng J., Wong K.H., Di L., Wong A.H., Xu X, & Deng C.X. (2021). Cisplatin prevents breast cancer metastasis through blocking early EMT and retards cancer growth together with paclitaxel. Theranostics, 11(5), 2442-2459.

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Lentiviral Transduction for S100A14 Overexpression

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Lentiviral vector (pLVX‐IRES‐Neo or pLVX‐IRES‐Neo‐S100A14) and packaging plasmids (pCMV Δ8.91, VSVG, and PLP2) were co‐transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The recombinant lentiviruses were harvested 48 hours after transfection, and subsequently pre‐cleaned with a 3000 g centrifuge and 0.45 μm filtration (Millipore, Billerica, MA, USA). For lentiviral transduction, 2 × 10⁵ cells/well were seeded into six‐well culture plates and infected the following day with lentiviruses plus 5 μg/mL Polybrene (Sigma‐Aldrich, USA). After the cells were infected with the lentivirus, 650 μg/mL geneticin was continuously added to the culture for two weeks. Overexpressing S100A14 stable clones were then obtained for further experiments.

Ding F., Wang D., Li X., Yang L., Liu H., Cui W., Liu Z, & Che Y. (2018). Overexpression of S100A14 contributes to malignant progression and predicts poor prognosis of lung adenocarcinoma. Thoracic Cancer, 9(7), 827-835.

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Lipoprotein Fractionation and ApoF Stability

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Plasma (250–500 μl) was fractionated on tandem Superose 6 columns, and 1 ml fractions were collected and assayed for their ApoF content by ELISA. As previously reported (9 (link)), two peaks of ApoF are observed—one coeluting with LDL and a second HDL-associated fraction with an apparent molecular mass of 470 kDa. This profile is stable for at least 2 months and for several freeze-thaw cycles when plasma is stored at −80°C. However, ApoF on LDL in plasma held at −80°C dissociates over time. At 26 weeks, approximately 20% of LDL-associated ApoF dissociates and is recovered in a novel ∼96 kDa peak. In long-term stored plasmas, very little ApoF remains on LDL. ApoF in the 470 kDa peak appears to be stable during storage. When analyzing the distribution of ApoF in frozen plasma samples, the amount of ApoF associated with LDL was taken as the sum of ApoF recovered in the LDL and 96 kDa peaks. Also, frozen plasma often contains particulate material due, in part, to VLDL aggregation. Particulates were removed before chromatographic analysis by 0.45 μm filtration (Millipore, Burlington, MA). Filtering did not remove detectable ApoF. Plasma cholesterol was reduced by <10%. However, this step did remove up to 50% of sample TG. During subsequent Superose 6 chromatography of filtered sample, the loss of cholesterol, TG, or ApoF was <10%.

Morton R.E, & Mihna D. (2022). Apolipoprotein F concentration, activity, and the properties of LDL controlling ApoF activation in hyperlipidemic plasma. Journal of Lipid Research, 63(2), 100166.

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Quantifying Organic Acids in Wine

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The organic acids were extracted following the methods reported by (Han et al., 2019) with modifications. Briefly, 5 mL samples were mixed with K4[Fe (CN)6]•3H2O (2 mL, 10.6%) and ZnSO4 (2 mL, 30%). Then, the mixture was diluted with pure water to 100 mL. After precipitation for 15 min, the collected supernatant was centrifuged at 6500×g at 4°C for 15 min. Finally, centrifuged solution was successively filtered through C-18 column and 0.45 μm filtration (Millipore, Bedford, MA, USA) prior to the analysis.
The identification and quantification of individual organic acids (tartaric acid, malic acid, lactic acid, acetic acid, succinic acid, oxalic acid and pyruvic acid) in wine samples from different vintages were performed by High Performance Liquid Chromatography (HPLC). The HPLC system was equipped with a Waters 1525 Binary HPLC pump, Waters 2707 autosampler injector, CTO-20AC oven, DGU-20A5R degasser and Waters 2489 ultraviolet-visible detector. All organic acids were separated using a C18 column (3.9×150 mm, 5 µm, Agilent) with column oven temperature of 30°C and the detector was set at 210 nm. The mobile phase was 0.02 mol L 1 NaH2PO4 and the pH was adjusted to 2.7 with phosphoric acid. The flow rate was 1 mL min 1 and total run time was 20 min. All samples injected into the chromatograph system was 10 µL.

Ding Y., Ma Y., Li S., Liang J., Xi X., Wang J., Sun J., Yu H., & Guo S. (2020). Characterization of Biochemical Compositions, Volatile Compounds and Sensory Profiles of Cabernet Sauvignon Wine in Successive Vintages (2008-2017). American journal of biochemistry & biotechnology/American journal of biochemistry and biotechnology, 16(3), 380-391.

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