Quantification of lysosomal pH was determined using dextran-conjugated Lysosensor Yellow/Blue (Fisher Scientific). About 3,000 cells were seeded in multiple wells of black, clear bottom tissue culture treated 96 well plates and grown to ~90% confluency (~24 hours). A 96 well plate was divided into 4 sectors (1) triplicated each pH standard wells for WT cell (2) triplicated each pH standard wells for PSEN1 KO cell, (3) WT cells w/ or w/o treatment, (4) PSEN1 KO cells w/ or w/o treatment. After that 2 ul of 0.05 mg/ml Lysosensor Yellow/Blue-dextran was added and incubated for 12 hours at 37°C with 5% CO2. The standard curve generation was performed according to the protocol established by Diwu et al. [71 (link)]. Briefly, cells were treated with 10 uM monensin and 10 uM nigericin in MES buffer (5 mM NaCl, 115 mM KCL, 1.3 mM MgSO4, 25 mM MES), with the pH adjusted to a range from 3.5–7.0 for 10 min after Lysosensor Yellow/Blue-dextran incubation. Cells were washed 3 times with HBSS and then read in a Wallac Victor 2 fluorimeter (Perkin Elmer) with excitation at 355 nm. The ratio of emission 440 nm/535 nm was then calculated for each sample. All readings were triplicated, and the average values were used for calculations. The pH values were determined from the standard curve generated via the pH calibration samples.
Wallac victor 2 fluorimeter
Manufactured by PerkinElmer
Sourced in United States, Italy
The Wallac Victor 2 is a fluorimeter designed for high-performance fluorescence detection. It provides accurate and reliable measurement of fluorescent signals in various microplate-based applications.
Automatically generated - may contain errors
4 protocols using wallac victor 2 fluorimeter
1
Lysosomal pH Quantification via Dextran-Conjugated Lysosensor
2
Lysosomal pH Measurement using LysoSensor
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Measurement of lysosomal pH was determined using dextran conjugates LysoSensor Yellow/Blue DND-160 (Invitrogen, L7545) [26 ]. In brief, cells were trypsinized, harvested (1 × 106 cells/ml), and loaded with 1 mg/ml LysoSensor-dextran for 1 h at 37°C with 5% CO2. The cells were then washed 3 times and aliquoted into a black 96-well microplate and pH calibration was performed according to the protocol established by Wolfe et al [26 ]. In brief, cells were treated with 10 mM monensin (Sigma-Aldrich, M5273) and 10 mM nigericin (Sigma-Aldrich, N7143) in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (5 mM NaCl, 115 mM KCl, 1.3 mM MgSO4, 25 mM MES [Sigma-Aldrich, M3671]), with the pH adjusted to a range from 3.5–7.0. The samples were read in a Wallac Victor 2 fluorimeter (Perkin Elmer, Norwalk, CT, USA) with excitation at 355 nm. The ratio of emission at 440/535 nm was then calculated for each sample. The pH values were determined from the linear standard curve generated via the pH calibration samples.
3
Lysosomal pH Measurement with Peptide
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Following the addition of 250 μg of LysoSensor Yellow/Blue dextran treatments, cells were incubated for 24 hours. The samples were then read in a Wallac Victor 2 fluorimeter (PerkinElmer) with an excitation at 355 nm. The ratio of emission 440 nm/535 nm was then calculated for each sample. The pH values were determined from the standard curve generated via pH calibration samples. To measure lysosomal pH in presence of peptide, we directly added 5 μM peptide to the cells for 24 hours at 37°C, wash out the peptide containing medium (three times), and then added 250 μg of LysoSensor Yellow/Blue dextran for 24 hours at 37°C.
4
Comprehensive Bioactive Compound Analysis
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Total polyphenol, flavonoid, tannin and Free amino acids content were determined by using a Cary 50 Bio spectrophotometer (Varian Inc., Turin, Italy). The same apparatus was also used for DPPH, ABTS and FRAP assays, and for the measurement of superoxide radical scavenging, hyaluronidase and elastase inhibition activities. Protein and carbohydrate contents were determined by using a PowerWave-HT BIO-TEK® (BIO-TEK instruments Inc., Vermont, VT, USA) microplate UV spectrometer. The same apparatus was also used for the measurement of tyrosinase and collagenase inhibition. Fluorescence for ORAC assay was measured by using a Wallac Victor2 fluorimeter (Perkin-Elmer Life Science, Monza, Italy).
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