Polyethylene nano/microplastics (PE N/MPLs) 200 nm–9900 nm size range (Cospheric LLC, Santa Barbara, CA, USA) were resuspended in 1% Tween20 (Dako, Agilent Technologies, Santa Clara, CA, USA) solution to obtain a 100 mg/mL stock, following the manufacturer’s directions. Working solutions were made by diluting the stock solution with ultrapure distilled H2O so that the final concentration of Tween20 in the culture media was maintained below 0.01% to minimise the surfactant-mediated cytotoxicity [72 (link)]. Stocks and working solutions were thoroughly vortexed before use. PE N/MPLs were used at 25 and 250 μg/mL in cell culture assays. After excluding that matching volumes of Tween20 alone could have effects on cells in different experimental settings, a vehicle control containing the same amount of Tween20 as that of the highest concentration of PE working solution was employed.
Tween 20
Manufactured by Agilent Technologies
Sourced in United States
Tween 20 is a non-ionic detergent commonly used in various laboratory applications. It is a polyoxyethylene sorbitan monolaurate that helps solubilize and stabilize proteins and other biomolecules. Tween 20 is a versatile reagent that can be used in a wide range of scientific procedures, including immunoassays, Western blotting, and cell culture.
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Lab products found in correlation
Tris buffered saline (tbs), by Agilent Technologies (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Menadione, by Merck Group (1 mentions)
Fitc concanavalin a, by Merck Group (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Mueller hinton agar (mha), by BD (1 mentions)
Mueller hinton broth (mhb), by BD (1 mentions)
Xtt salt, by Merck Group (1 mentions)
Pseudomonas aeruginosa atcc 9027, by American Type Culture Collection (1 mentions)
Staphylococcus aureus atcc 6538, by American Type Culture Collection (1 mentions)
Protein blocking solution, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
5 protocols using tween 20
1
Polyethylene Nano/Microplastics Exposure
2
Biofilm Inhibition Assay Protocols
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PLGA (50 : 50) (mol. wt 30 000–60 000 g mol−1), xylitol, polyvinyl alcohol (PVA), Pluronic F-125, PBS phosphate buffer saline (PBS), XTT salt, menadione and concanavalin A-FITC were purchased from Sigma-Aldrich Chemicals Co. Ltd. (St. Louis Missouri, USA). Acetonitrile, methanol and dichloromethane were purchased from J. T. Baker Avantor performance Materials, Inc (Malaysia). Tween 20 was purchased from Agilent Technologies, USA. Two biofilm-forming reference bacterial strains were used, i.e., Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 9027) and they were obtained from American Type Culture Collection (Erbetta, #23). A polymicrobial biofilm was formed by mixing equal ratios of both bacterial strains in the same culture. Mueller–Hinton agar (MHA) and Mueller–Hinton broth (MHB) were purchased from Becton, Dickinson and Company. Methanol, acetone and glacial acetic acid were purchased from R & M Chemicals Ltd (United Kingdom); ethanol was purchased from John Kollin Corporation (United Kingdom). A LIVE/DEAD™ BacLight™ Bacterial Viability Kit for microscopy and quantitative assays was purchased from Thermo-Fisher scientific, USA.
3
Immunohistochemical Analysis of YBX1 in Colon Cancer
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Colon cancer tissue microarray comprising matched normal adjacent and various clinical stages of colon carcinoma tissue was obtained from US Biomax Inc. [Rockville, MD]. Hematoxylin-eosin [H&E] was performed using routine methods. The tissue microarray were blocked with protein blocking solution [Dako Corp.] for 30 min. All subsequent staining steps were performed using the Dako FLEX SYSTEM on an automated Immunostainer. Incubations were done at room temperature and Tris buffered saline plus 0.05% Tween 20, pH 7.4 [Dako Corp] was used for all washes and diluents. Thorough washing was performed after each incubation. Anti-YBX1 primary antibody was used to detect YBX1 localization. Horseradish peroxidase conjugated secondary antibody was then used, followed by addition of the chromogen, which produced a brown precipitate at the site of secondary antibody binding. The Aperio whole slide digital imaging system was used for imaging. The system imaged all slides at 20X.
4
Immunohistochemical analysis of PRMT5 in cancer
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Pancreatic and colon cancer tissue microarrays with matched normal adjacent controls were acquired from US Biomax Inc. The tissue microarrays were blocked using protein-blocking solution (Dako Corp.) for 30 min. All subsequent staining steps were performed using the Dako FLEX SYSTEM and an automated Immunostainer. Incubations were carried out at room temperature and Tris buffered saline containing 0.05% Tween 20, pH 7.4 (Dako Corp.) was used for all the washes and diluents. Anti-PRMT5 primary antibody (Abcam; ab109451) was used to detect PRMT5 localization. Horseradish peroxidase-conjugated secondary antibody was then used, followed by addition of the chromogen, which formed a brown precipitate at the binding site of secondary antibody. Imaging was done using Aperio whole slide digital imaging system. The system imaged all slides at 20X magnification.
5
Immunohistochemical Analysis of Frozen Tissues
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Biopsy specimens frozen in liquid nitrogen and kept at À80 C were cut into sections (10 mm thick) and fixed in 1% methanol-free paraformaldehyde (Sigma Aldrich, Darmstadt, Germany). Blocking was performed with 5% normal goat serum in tris-buffered saline supplemented with 0.05% Tween-20 (Dako, Glostrup, Denmark). Thereafter, sections were incubated with primary antibodies; details can be found in Table 1. After rinsing of unbound antibody, the sections were incubated with appropriate secondary antibodies raised in goat (dilution, 1:600) for 1 hour at room temperature, as shown in Table 1. Finally, sections were counterstained with DAPI (300 nmol/L; Life Technologies, Carlsbad, CA) and further processed for image analysis. Fluorescent signals were visualized using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany). Human spleen and tonsil were used as positive controls. Omission of primary antibody served as negative control.
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