Cellular lysosomal environment was mimicked to determine stress induced on fluorescence through lysosomal material exposure. The citric acid (>99.5%, ACS Reagent, Sigma-Aldrich)–simulated lysosome chelator buffer at pH 2.5 to 5.0 was created and used as the solvent for QSH and PS. Controls contained pH 7.4 Dulbecco’s Phosphate-Buffered Saline (DPBS) buffer solutions. More specifically, stock solutions of 0.25 and 0.19 mM solutions of sodium citrate monobasic (Anhydrous, Sigma-Aldrich) and dibasic (sesquihydrate, Sigma-Aldrich), respectively, were made. Stock solutions of 50 and 20 mM citric acid stock solution were also made in separate vials. Then, six solutions of equal concentrations of 10 nM QSH were made in either sodium citrate monobasic/dibasic with citric acid. To achieve the desired pH of 2.5, 3.0, 3.5, 4.5, or 5.0, pH was adjusted by combination of dibasic or monobasic sodium citrate stock solution with small aliquots of citric acid solutions. For size analysis, Zetasizer (Malvern) DLS measurements were obtained. Here, samples were diluted in situ in solvents of desired pH and measurements were obtained immediately after. Fluorescent plate readings were run in triplicate of 100 μl of solutions applied to wells of a 96-well plate system. Fluorescence was taken with 580- or 525-nm excitation and 595- or 620-nm emission, respectively, for QSH or PS, using a Tecan M200 plate reader. To check for Cd2+ core leakage, 10 nM QSH and PS were analyzed for fluorescence in phosphate-buffered solution (PBS), water, and simulated lysosomal buffer at pH 2.5, 4.5, and 5.0 at 0- and 24-hour exposure. For each time point, samples were collected and centrifuged at 15,000g for 20 min through an Amicon Ultra 10-kDa filter to separate possible cations from QSH. Filtrate was then analyzed for free cadmium content using a PerkinElmer atomic absorption spectrometer with a cadmium hollow cathode lamp with a wavelength of 288.65 nm. Flow rate was adjusted to 4 ml/min, and samples were run in triplicate.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.