0.45 μm sterile filter

Manufactured by Merck Group

Sourced in United States, Germany

The 0.45-μm sterile filter is a laboratory equipment designed to remove particulates, bacteria, and other contaminants from liquid samples. It features a 0.45-micron pore size membrane that effectively filters out microorganisms while allowing the passage of the desired liquid.

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Lab products found in correlation

Plko 1 puro plasmid, by Addgene (1 mentions) Pltr g, by Addgene (1 mentions) Polyfect transfection reagent, by Qiagen (1 mentions) Pcmv dr8.2 dvpr, by Addgene (1 mentions) Multichannel pipette, by Eppendorf (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Trypsin, by Merck Group (1 mentions) P28s rotor, by Hitachi (1 mentions) Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Sterile filters (29 citations) 0.45-μm pore-size sterile disk filters (2 citations)

8 protocols using 0.45 μm sterile filter

Lentiviral Transduction of shRNA in HEK293T Cells

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HEK293T cells
were seeded in a 10 cm dish at 30% confluence 1 day prior to transfection
with 4 μg of pLKO.1 puro plasmid (Addgene no. 8453) for shRNA
expression, 1 μg of pLTR-G (Addgene no. 17532) envelope plasmid,
and 3 μg of pCMV-dR8.2 dvpr (Addgene no. 8455) package plasmid
together with 40 μL of PolyFect transfection reagent (Qiagen).
The medium was replaced 12 h after transfection. After 48 h, viral
particles were harvested from the culture medium and filtered with
a 0.45-μm sterile filter (Millipore). Cells were transduced
with a lentivirus for 48 h and subsequently screened with 1.0 μg/mL
puromycin. The shRNA sequences are listed in Table S4.

He X., Yuan J., Gao Z, & Wang Y. (2023). Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing. Journal of the American Chemical Society, 145(39), 21646-21660.

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Virus Isolation from Aquatic Animal Tissues

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Collected tissues were homogenized in phosphate-buffered saline (PBS) and clarified by centrifugation at 10,000× g for 5 min. The supernatants were filtered through a 0.45 μm sterile filter (Millipore, Burlington, MA, USA) and used to infect cell lines.
Different aquatic animal cell lines, Chinese giant salamander thymus cell (GSTC), Epithelioma Papulosum Cyprini (EPC), and Siniperca chuatsi skin cell (SCSC), which were preserved in our lab, were used in virus isolation and infection. The cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS) at 25 °C, except the SCSC cells were cultured in L15 medium with 10% FBS. For virus isolation, monolayers of these cells were inoculated with the above tissue homogenates and incubated at 25 °C. The cells were harvested when advanced cytopathic effects (CPE) were observed, and the supernatant was used for the next round of infection until a stable CPE was obtained. Finally, the infected cells were collected and used as virus stocks after being frozen and thawed. The virus titers were measured by using a 50% tissue culture infectious dose (TCID₅₀) assay as described previously [5 (link)].

Yu X.D., Ke F., Zhang Q.Y, & Gui J.F. (2023). Genome Characteristics of Two Ranavirus Isolates from Mandarin Fish and Largemouth Bass. Pathogens, 12(5), 730.

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Quantification of Amino Acids in Homogenates

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Measurement was performed according to Grimm et al. [29 (link)]. Briefly, 10μl of homogenates was applied onto a solvinert 96well plate with a 0.45μm sterile filter at the bottom (Millipore, Schwalbach, Germany). Samples were further incubated in a 5% phenylisothiocyanate solution diluted in C₂H₅OH/H₂O/pyridine (1:1:1; v/v/v) for 20’. Samples were extracted using 5mM ammonium acetate buffer (300μl) in methanol using a multichannel pipette (Eppendorf, Germany) into a 1ml 96well deep well plate (Nunc, Langenselbold, Germany) and further diluted with 600μl of 5mM ammonium acetate dissolved in CH₃OH/H₂O, which also served as the only running solvent.

Wittmann A., Grimm M.O., Scherthan H., Horsch M., Beckers J., Fuchs H., Gailus-Durner V., Hrabě de Angelis M., Ford S.J., Burton N.C., Razansky D., Trümbach D., Aichler M., Walch A.K., Calzada-Wack J., Neff F., Wurst W., Hartmann T, & Floss T. (2016). Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice. PLoS ONE, 11(10), e0164298.

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Rotavirus Isolation and Culture in MA104 Cells

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Five samples were also culture-adapted to MA104 cells as previously described with some modification^{28 (link),29 (link)}. Briefly, 1 ml of a 10% stool suspension was filtered through a 0.45-μm sterile filter (Merck Millipore, Billerica, USA) and activated in the presence of 15 μg of trypsin (Sigma‐Aldrich Cat.No. 85450 C, St. Louis, USA) for 1 h at 37 °C. Confluent monolayers of MA104 cells in roller tubes were then inoculated with stool suspensions for 1 h, unabsorbed virus was removed, and 2 ml of DMEM containing neomycin (Gibco Laboratories, California, USA) and 40 μg of trypsin were added. Tubes were harvested when cytopathic effect was complete or if no CPE is visible after 4 days, the tubes were frozen-thawed (−70 °C to room temperature) for three times before the next passage, freeze-thawed cell lysates treated with trypsin as described above for preparation of stool supernatants and was performed subsequent passages as described above. After four passages, lysates were tested for RV by real-time RT-PCR and EIA.

Li J.S., Cao B., Gao H.C., Li D.D., Lin L., LI L.L., Liu N, & Duan Z.J. (2018). Faecal shedding of rotavirus vaccine in Chinese children after vaccination with Lanzhou lamb rotavirus vaccine. Scientific Reports, 8, 1001.

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Purification and Titration of Respiratory Syncytial Virus

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The viral samples were inoculated onto HEp-2 cells with 70%–80% confluence and incubated for 3 days at 37°C under 5% CO₂. After the syncytia formation, the cells were scraped off and centrifuged at 3000 rpm for 10 min at 4°C to remove cellular debris. Supernatants were pooled, filtered through a 0.45 μm sterile filter (Merck Millipore, Carolina, USA) and purified by ultracentrifugation on sucrose cushion gradient (10% sucrose, Sigma) at 17,000 rpm in P28S rotor (Hitachi, Japan) for 2 h at 4°C. The pellet was suspended in 300 μl 10% sucrose and divided into aliquots and stored at −80°C. The infectivity of the RSV was titrated using the method of immunoplaque assay as described previously [9 (link)].

Xu M., Jiao Y.Y., Fu Y.H., Jiang N., Zheng Y.B., Yan Y.F., Zhang M., Zheng Y.P., Zhu W.Y., Peng X.L, & He J.S. (2018). Construction and Characterization of a Recombinant Human Respiratory Syncytial Virus Encoding Enhanced Green Fluorescence Protein for Antiviral Drug Screening Assay. BioMed Research International, 2018, 8431243.

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Quantifying Ion Release from Bioactive Glass

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To quantify the ion release from BG particles, the respective BGs were incubated in UltraPure DNase/RNase-Free Distilled Water (Life Technologies, Darmstadt, Germany) at a concentration of 10 mg/ml for 24 h (representing the preconditioning phase) as well as for 1, 7, 14 and 24 days. After incubation, the supernatant was collected and filtered through a 0.45 μm sterile filter (Merck, Darmstadt, Germany). The samples were acidified with 50 μl concentrated nitric acid per 10 ml sample. To avoid interference of various ions present in the CCM the measurement had to be performed in distilled water. The release of Ca, magnesium (Mg), Na, phosphate (P) and Si was measured via inductively coupled plasma optical emission spectroscopy using an Agilent 720 ICP-OES instrument (Agilent, Santa Clara, USA). All samples were measured in triplicates.

Schmitz S.I., Widholz B., Essers C., Becker M., Tulyaganov D.U., Moghaddam A., Gonzalo de Juan I, & Westhauser F. (2020). Superior biocompatibility and comparable osteoinductive properties: Sodium-reduced fluoride-containing bioactive glass belonging to the CaO–MgO–SiO2 system as a promising alternative to 45S5 bioactive glass. Bioactive Materials, 5(1), 55-65.

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JMTV Isolation in Mammalian and Insect Cells

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Processed tick homogenates which were positive for JMTV RNA by qRT-PCR were inoculated onto semi-confluent monolayers of Vero, Vero E6, BHK21 and C6/36 cells. The cells were cultured in DMEM or DMEM/F12 containing 10% heat-inactivated fetal bovine serum (FBS), and maintained at 37°C in 5% CO₂ or at 27°C. Briefly, the specimens were subjected to centrifugation, and the supernatant was passed through a sterile 0.45-μm filter (Merck Millipore, USA). Then, the filtrates (100 μL) were inoculated onto cells in six-well plates. After cell adsorption for one hour, 2 mL of DMEM or DMEM/F12 supplemented with 5% fetal bovine serum, l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin were added and the cells were incubated under the same conditions for 7–10 days. Cells were monitored daily for cytopathic effects and detected for viral RNA by qRT-PCR on day 6–8 post-infection. After another two blind passages, the supernatants were harvested and preserved at −80°C until further analysis. Virus isolation was performed at a BSL-2 laboratory in School of Public Health, WeiFang Medical University.

Pang Z., Jin Y., Pan M., Zhang Y., Wu Z., Liu L, & Niu G. (2022). Geographical distribution and phylogenetic analysis of Jingmen tick virus in China. iScience, 25(9), 105007.

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Isolation of Dengue Virus from Tick Samples

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Tick homogenates which were positive for DTV viral RNA by qRT-PCR were subjected to centrifugation, and the supernatant was passed through a sterile 0.45 μm filter (Merck Millipore, Billerica, MA, USA). Then, the filtrates were subjected to virus isolation using the cell line BHK–21 cultured in six-well plates. Briefly, 100 μL of sample was inoculated onto a monolayer of BHK–21 cells at 37 °C in 5% CO₂ conditions for 7 days. Cells were inspected daily and tested for the presence of DTV RNA by qRT–PCR on day 6–8 post-infection. After three additional blind passages, the supernatants were harvested and cryopreserved at -80 °C until further analysis. Virus isolation was performed at a BSL–2 laboratory in School of Public Health, WeiFang Medical University.

Wang A., Pang Z., Liu L., Ma Q., Han Y., Guan Z., Qin H, & Niu G. (2021). Detection and Phylogenetic Analysis of a Novel Tick-Borne Virus in Yunnan and Guizhou Provinces, Southwestern China. Pathogens, 10(9), 1143.

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