W4502

Manufactured by Merck Group

Sourced in United States

W4502 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to assist in various scientific experiments and analytical procedures carried out in a controlled laboratory environment.

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Lab products found in correlation

22 protocols using w4502

Fission Yeast Growth and Genetic Protocols

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Standard methods for S. pombe growth and genetics were used [47 ]. Cells were typically grown in exponential phase in liquid YE5S medium at 25 °C with shaking for 18–24 h.
Methyl benzimidazol-2-yl-carbamate (MBC) was dissolved in dimethyl sulfoxide to make a 10 mg/mL stock solution. Geneticin (G418) was dissolved in pure water (Sigma-Aldrich #W4502) to make a 50 mg/mL stock solution. ClonNAT was dissolved in pure water (Sigma-Aldrich #W4502) to make a 200 mg/mL stock solution.

Flor-Parra I., Iglesias-Romero A.B, & Chang F. (2018). The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast. Current biology : CB, 28(11), 1681-1691.e4.

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Nanoparticle Tracking Analysis of Exosomes

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Exosome samples were analyzed as previously described (21 (link)). Briefly, exosomes isolated from 100 μl plasma were resuspended in 100 μl PBS and diluted 1:10,000 in particle-free water (W4502, Sigma). Exosomes isolated from 10⁷ cells were resuspended in 50 μl PBS and diluted 1:10,000 in particle-free water. After vortexing, the diluted samples were injected into the NTA LM-10 system continuously using a syringe pump. Particles were acquired by the machine, and data were analyzed with NTA particle analysis software.

Li W., Deng M., Loughran P.A., Yang M., Lin M., Yang C., Gao W., Jin S., Li S., Cai J., Lu B., Billiar T.R, & Scott M.J. (2020). LPS Induces Active HMGB1 Release From Hepatocytes Into Exosomes Through the Coordinated Activities of TLR4 and Caspase-11/GSDMD Signaling. Frontiers in Immunology, 11, 229.

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Monomeric UHRF1 Visualization by AFM

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A 20 nM solution of UHRF1 (25 mM HEPES pH 7.4, 100 mM NaOAc, 1 mM DTT) was mixed with or without HeDNA (5 μM) and deposited on freshly peeled mica, immediately rinsed with water (Sigma #W4502), and dried with nitrogen gas before imagining. All images were acquired on the same day as the deposition. Images were collected on an MFP3D Atomic Force Microscope (Asylum Research Oxford Instruments using the following parameters: scan rate 1 Hertz, scan size 1 μM x 1 μM, image resolution 1024 x 512. Images were collected in intermittent contact mode (AC mode) using AFM probes from NanoSensor (PPP-FMR, force constant = 2.8 N/m). Images were analyzed using the Asylum Research AFM software package. The images were flattened to a second-degree polynomial to account for surface warping artifacts and volume analysis was performed using built-in particle analysis (a more detailed review of this methodology can be found here Ratcliff and Erie, 2001 (link)). Volume distributions were plotted to a peak fit model and visualized using Origin 6.1 (origin labs). The fact we could only identify a single volume species indicates monomeric UHRF1; the kD (data not shown) we calculated from AFM volume is also in agreement with monomeric UHRF1).

Harrison J.S., Cornett E.M., Goldfarb D., DaRosa P.A., Li Z.M., Yan F., Dickson B.M., Guo A.H., Cantu D.V., Kaustov L., Brown P.J., Arrowsmith C.H., Erie D.A., Major M.B., Klevit R.E., Krajewski K., Kuhlman B., Strahl B.D, & Rothbart S.B. (2016). Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1. eLife, 5, e17101.

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RNA to cDNA Conversion Protocol

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1–2 μg of RNA were treated with Amplification Grade DNase I (18068015, Invitrogen) by following the manufacturer’s guidelines. cDNA was synthesized with M-MLV reverse transcriptase (28025013, Invitrogen) in the presence of Random Primers (48190011, Invitrogen), dNTPs (10297018, Invitrogen), and RNaseOUT recombinant ribonuclease inhibitor (10777019, Invitrogen), by using a Veriti Dx thermal cycler (Applied Biosystems). RT conditions involved 10 min at 25°C, 3h at 37°C and 15 min at 70°C. The resulting cDNA was diluted 10-fold in nuclease-free water (W4502, Sigma-Aldrich).

Lachiondo-Ortega S., Rejano-Gordillo C.M., Simon J., Lopitz-Otsoa F., Delgado T.C., Mazan-Mamczarz K., Goikoetxea-Usandizaga N., Estefanía Zapata-Pavas L., Garcıía-del Río A., Guerra P., Peña-Sanfélix P., Hermán-Sánchez N., Al-Abdulla R., Fernandez-Rodríguez C., Azkargorta M., Velázquez-Cruz A., Guyon J., Martín C., Zalamea J.D., Egia-Mendikute L., Sanz-Parra A., Serrano-Maciá M., González-Recio I., Gonzalez-Lopez M., Martínez-Cruz L.A., Pontisso P., Aransay A.M., Barrio R., Sutherland J.D., Abrescia N.G., Elortza F., Lujambio A., Banales J.M., Luque R.M., Gahete M.D., Palazón A., Avila M.A., Marin J.J., De S., Daubon T., Díaz-Quintana A., Díaz-Moreno I., Gorospe M., Rodríguez M.S, & Martínez-Chantar M.L. (2024). SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer. Cell reports, 43(3), 113924.

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Exosome Characterization by Nanoparticle Tracking

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The exosome samples were analysed, as previously described (Ouyang et al, 2016 (link)). Briefly, exosome samples in PBS (protein concentration of ∼1 μg μl⁻¹, as measured by BCA assay) were diluted 1 : 500 in particle-free water (W4502, Sigma, St Louis, MO, USA). After vortexing, the diluted samples were injected into the NTA LM-10 system continuously using a syringe pump. Particles were acquired by the machine and data were analysed with NTA particle analysis software. Exosome particles were normalised to protein concentration.

Yang M.Q., Du Q., Varley P.R., Goswami J., Liang Z., Wang R., Li H., Stolz D.B, & Geller D.A. (2017). Interferon regulatory factor 1 priming of tumour-derived exosomes enhances the antitumour immune response. British Journal of Cancer, 118(1), 62-71.

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Nanoparticle Tracking Analysis Protocol

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MVs or EXs were diluted to an appropriate level (1,000- to 5,000-fold
dilution) with 0.1 µM filtered water (W4502, Sigma), which has been
validated in our NTA LM-10 system to be particle-free. This dilution ensured
that at least 20 particles would be observed in the objective view field per
second. The diluted samples were continuously injected into the view field by a
syringe pump. Particles were individually recorded and tracked by the
high-definition CCD camera for 1 min. Each measurement was repeated three times.
Installed NTA particle analysis software was used to analyze all frames
containing particles captured by the camera.

Ouyang Y., Bayer A., Chu T., Tyurin V.A., Kagan V.E., Morelli A.E., Coyne C.B, & Sadovsky Y. (2016). Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity. Placenta, 47, 86-95.

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Quantitative PCR Assay for Detection

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Each 15 µl qPCR reaction mixture contained 1 × TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific Inc.), 0.1 mg/ml bovine serum albumin (BSA) (Sigma–Aldrich), 250 nM of each primer, 100 nM of each TaqMan probe, 1 × TaqMan™ Exogenous Internal Positive Control (VIC-TAMRA labelled) Reagents (Thermo Fisher Scientific Inc.) and 2 µl template DNA. A negative control was included in each run, which consisted of nuclease-free water (W4502; Sigma–Aldrich). The qPCR program consisted of 2 min at 50 °C, 3 min at 95 °C, followed by 45 cycles of 95 °C for 3 s and 60 °C for 30 s. Amplification was performed in a CFX Opus 96 Real-Time PCR Instrument (Bio-Rad Laboratories Inc., Hercules, CA, USA) and analysed by the CFX Maestro Software version 2.0 (Bio-Rad Laboratories Inc.) with default settings. Samples for which a copy number equal to or above the limit of detection (LOD) was calculated from the qPCR assays were considered as positive.

Frosth S., Eriksson H.K, & Rosander A. (2023). Development of a multiplex quantitative PCR assay for simultaneous detection of Treponema phagedenis, Treponema pedis, Treponema medium, and ‘Treponema vincentii’ and evaluation on bovine digital dermatitis biopsies. Veterinary Research Communications.

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Endosomal and Lysosomal Dynamics in SNX5 KO Cells

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Wild-type and SNX5^KO HeLa/GFP-LC3 cells were cultured in Opti-MEM I Reduced Serum Medium supplemented with 5% FBS (normal medium) in Nunc Lab-Tek II 4-well glass chamber slides overnight. Hypotonic medium was made by mixing fresh normal medium with sterile water (Sigma-Aldrich W4502) at a ratio of 20:80 (vol/vol). Cells were rinsed three times with DPBS (Sigma-Aldrich D8537) and then cultured in fresh normal medium or hypotonic medium for 1 h. Cells were fixed and immunofluorescence microscopy was performed using the protocol described in the section “Fluorescence microscopy and image analyses” and stained for the early endosomal marker EEA1 and the lysosomal marker LAMP1.

Dong X., Yang Y., Zou Z., Zhao Y., Ci B., Zhong L., Bhave M., Wang L., Kuo Y.C., Zang X., Zhong R., Aguilera E.R., Richardson R.B., Simonetti B., Schoggins J.W., Pfeiffer J.K., Yu L., Zhang X., Xie Y., Schmid S.L., Xiao G., Gleeson P.A., Ktistakis N.T., Cullen P.J., Xavier R.J, & Levine B. (2020). Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity. Nature, 589(7842), 456-461.

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Cell Viability Assay with Molecular Water

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Cell viability was detected using the Cellrix® Viability Assay Kit (B1007-500, Cellrix). HEK293A cells (5 × 10³ cells per well) were seeded in the 96-well plates. After culturing for 24 h, the medium was removed, and the cells were exposed to different concentrations of molecular water (W4502, Sigma) and LPP100-rGOQDs. After 24 h of incubation, the Cellrix® Viability Assay Kit was added and incubated additional 1 h. The absorbance was measured at a test wavelength of 450 nm and a reference wavelength of 600 nm by the GloMax™ Multi Reader (9301-010, Promega).

Lee J.W., Kwak J.H., Kim J., Jang Y.K., Han J.T., Kim T.J., Hong K.S., Jeong H.J, & Yang I.H. (2024). Highly emissive blue graphene quantum dots with excitation-independent emission via ultrafast liquid-phase photoreduction. RSC Advances, 14(16), 11524-11532.

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DNA Extraction from Swab Samples

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The swab tips were submerged in 1 mL of molecular grade water (W4502, Sigma, UK), and bacteria released by 10s manual agitation. DNA extraction from 200 μl of the water was performed using NucleoSpin® 96 soil kit (740787, Macherey-Nagel, Germany) using an EpMotion 5075vt (Eppendorf, USA), following manufacturer instructions, using lysis buffer SL1 with 150 μl Enhancer SX and eluted in 70 μl elution buffer.

Rodrigues R., Chung A.P., Mortensen M.S., Fernandes M.H., Monteiro A.B., Furfuro R., Silva C.C., Manso M.C., Sørensen S.J, & Morais P.V. (2021). Temporal oral microbiome changes with brushing in children with cleft lip and palate. Heliyon, 7(3), e06513.

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