W3500

Manufactured by Merck Group

The W3500 is a laboratory centrifuge designed for separating and concentrating samples. It features a compact and durable construction with a maximum speed of 3,500 RPM and a maximum RCF (Relative Centrifugal Force) of 1,697 x g. The W3500 is suitable for a variety of applications in research and clinical laboratories.

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Lab products found in correlation

S2770, by Merck Group (3 mentions) Sw32ti rotor, by Beckman Coulter (1 mentions) Collagenase type 1a, by Merck Group (1 mentions) Sodium nitroprusside, by Merck Group (1 mentions) Adenosine deaminase ada, by Merck Group (1 mentions) Carbamylcholine chloride carbachol, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)

6 protocols using w3500

Production and Infection of Lentiviral Particles

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For lentiviral particle production, 7.5 million low passage 293T/17 cells were seeded in 15 cm plates, incubated overnight and 2 h before transfection media was replaced. For each plate, 44 µg of pLKO.1-shRNA (Sigma), 13 µg of VSV-G envelope and 33 µg of delta 8.9 packaging vectors were mixed with 1125 µL of endotoxin-free water (Sigma, W-3500) and 125 µL of 2.5 M CaCl₂ (Sigma). After incubating the mix for 5 min in a rotor, 1250 µL of 2× HBS pH 7.2 (281 mM NaCl, 100 mM HEPES, 1.5 mM Na₂HPO₄) were added in a drop-wise manner. After 10min of incubation at room termperature, the precipitate was added dropwise to the cells and incubated for 30 h. Virus-containing supernatants were collected, filtered (0.22 µm) and centrifuged at 20,000rpm at 4°C for 2 h in a SW32Ti rotor (Beckman). Lentiviral pellets were resuspended in 80 µL of PBS and kept at −80°C.
For lentiviral infections, cells were incubated with concentrated lentivirus overnight and subcultured with fresh medium the following day. Forty-eight hours after infection, cells were selected with puromycin (1 µg/mL for U2OS, 2 µg/mL for HeLa) for 2 d.

Martín E., Vivori C., Rogalska M., Herrero-Vicente J, & Valcárcel J. (2021). Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation. RNA, 27(12), 1557-1576.

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Peptide-based Hydrogel Fabrication

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A commercially available peptide preparation in powder form was used as the source of the octapeptide gelator (FEFEFKFK, Phe-Glu-Phe-Glu-Phe-Lys-Phe-Lys). As part of this study we used peptide sourced from Cambridge Research Biochemicals (batch 32597) although we also verified the fabrication method using a second peptide source (Pepceuticals, UK). To form each precursor, a mass of between 7.5 and 18.75 mg peptide preparation was dissolved in 800 μL sterile water (W3500 Sigma), using a 3 min vortex step followed by centrifugation (3 min at 1000 rpm) and a 2 h incubation at 80 °C. After incubation, 0.5 M NaOH (S2770 Sigma) was added incrementally to the gels until optically clear. Gels were vortexed, buffered by addition of 100 μL 10× PBS (70011 Gibco), and incubated at 80 °C overnight. The resulting precursors could be stored at 4 °C until required.

Ashworth J.C., Thompson J.L., James J.R., Slater C.E., Pijuan-Galitó S., Lis-Slimak K., Holley R.J., Meade K.A., Thompson A., Arkill K.P., Tassieri M., Wright A.J., Farnie G, & Merry C.L. (2019). Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro. Matrix biology : journal of the International Society for Matrix Biology, 85-86, 15-33.

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Cultivation of Treponema pallidum

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TpCM-2 was prepared as indicated in Table 4 1 day prior to use. All solutions were made in cell culture-grade water (Sigma W3500) and filter sterilized. Fetal bovine serum (FBS) lots vary considerably in their ability to support T. pallidum survival and replication (16 (link), 38 (link), 51 (link)). FBS lots from several suppliers were therefore prescreened for efficacy (by comparison with previously tested lots), purchased in bulk, and heat inactivated at 56°C for 30 min prior to use. Samples of FBS lots that support the multiplication of T. pallidum are available upon request. The pH of the medium was adjusted to 7.5, and the mixture was then filter sterilized with 0.22-µm-pore-size polyethersulfone filters (Merck Millipore). The medium was then preequilibrated in a BBL GasPak jar in which a vacuum was drawn five times (house vacuum; ~12 to 18 µm Hg), and the jar was refilled with 5% CO₂–95% N₂ four times and a final time with 1.5% O₂–5% CO₂–93.5% N₂. The medium was then incubated overnight in a Forma model 3130 trigas incubator (Thermo Fisher) maintained at 34°C with 1.5% O₂–5% CO₂–93.5% N₂ (here referred to as the low-oxygen incubator). All subsequent steps in the incubation of T. pallidum cultures were carried out under these conditions.

Edmondson D.G., Hu B, & Norris S.J. (2018). Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum. mBio, 9(3), e01153-18.

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Hydrogel Precursor Synthesis Protocol

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The precursor and gel preparation method was followed as previously published⁵⁷. A commercially available peptide preparation in powder form was used as the source of the octapeptide gelator (Pepceuticals UK, FEFEFKFK, Phe-Glu-Phe-Glu-Phe-Lys-Phe-Lys). To form the precursor, a mass of 10 mg peptide preparation was dissolved in 800 μL sterile water (Sigma, W3500), using a 3 min vortex step followed by centrifugation (3 min at 1000 rpm) and a 2 h incubation at 80 °C. After incubation, 0.5 M NaOH (Sigma, S2770) was added incrementally to the gel until optically clear. The gel was vortexed, buffered by addition of 100 μL 10× PBS (Gibco, 70011), and incubated at 80 °C overnight. The resulting precursor could be stored at 4 °C until required.

Mendonca T., Lis-Slimak K., Matheson A.B., Smith M.G., Anane-Adjei A.B., Ashworth J.C., Cavanagh R., Paterson L., Dalgarno P.A., Alexander C., Tassieri M., Merry C.L, & Wright A.J. (2023). OptoRheo: Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems. Communications Biology, 6, 463.

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Neurotransmitter Receptor Agonist Protocols

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ATP (disodium salt), -meATP (lithium salt), adenosine deaminase (ADA), carbamylcholine chloride (Carbachol), collagenase (Type 1A), sodium nitroprusside, TEA (chloride salt) and TTX were obtained from Sigma-Aldrich (Gillingham, UK). Sterile water (W3500; Sigma Aldrich) was used in the luciferase assay. MRS2179 was obtained from Tocris-Cookson Ltd (Bristol, UK) and A-317491 was a gift from Abbvie Inc. (Illinois, USA).
Conotoxin GVIA was a gift from Professor Annette Dolphin (NPP, UCL). All drugs were made up in de-ionised, filtered and sterile water as stocks of either 10 mM or 100 mM, and stored at -20°C. Drugs were thawed just before use and diluted in a modified Krebs solution or Ringer's solution.

King B.F. (2021). P2X3 receptors participate in purinergic inhibition of gastrointestinal smooth muscle. Autonomic neuroscience : basic & clinical, 234.

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Octapeptide Hydrogel Precursor Preparation

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The precursor and gel preparation method was followed as previously published 57 (link) . A commercially available peptide preparation in powder form was used as the source of the octapeptide gelator (Pepceuticals UK, FEFEFKFK, Phe-Glu-Phe-Glu-Phe-Lys-Phe-Lys). To form the precursor, a mass of 10 mg peptide preparation was dissolved in 800 μL sterile water (Sigma, W3500), using a 3 min vortex step followed by centrifugation (3 min at 1000 rpm) and a 2 hour incubation at 80 °C. After incubation, 0.5 M NaOH (Sigma, S2770) was added incrementally to the gel until optically clear. The gel was vortexed, buffered by addition of 100 μL 10x PBS (Gibco, 70011), and incubated at 80 °C overnight. The resulting precursor could be stored at 4 °C until required.

Mendonca T., Lis-Slimak K., Matheson A.B., Smith M.G., Anane‐Adjei A.B., Ashworth J., Cavanagh R., Paterson L., Dalgarno P.A., Alexander C., Tassieri M., Merry C.L., & Wright A.J. (2022). OptoRheo: Simultaneousin situmicro-mechanical sensing and imaging of live 3D biological systems.

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