The WST-1 cell growth assay was carried out to determine the cytotoxic effect on PC-3 cells of Chlor, to select the concentration suitable for the in vitro sonodynamic treatment. Briefly, in 96-well culture plates (TPP, Trasadingen, Switzerland) 1.5 × 103 cells per well were seeded in 100 μL of culture medium in 8 replicates; after 24 h, the medium was removed and the cells incubated with experimental media of differing Chlor concentrations (5, 10, 50, 100 and 500 µM) obtained by diluting in DMEM a 50 mM Chlor DMSO solution. The WST-1 reagent (10 μL/100 µL) was added at 24, 48, and 72 h and the plates were incubated at 37 °C in 5% CO2 in air for 90 min. A microplate reader (Asys UV340, Biochrom, Cambridge, UK) was used to detect well absorbance at 450 and 620 nm (reference wavelength).
Asys uv340
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The Asys UV340 is a compact and versatile UV-Vis spectrophotometer. It features a dual-beam optical system and a wavelength range of 190 to 1100 nm. The instrument is designed for precise absorbance measurements and can be used for a variety of applications in life science research and analytical chemistry.
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Lab products found in correlation
Glutathione assay kit, by Merck Group (3 mentions)
Quant it protein assay kit, by Thermo Fisher Scientific (2 mentions)
Wst 1 reagent, by Merck Group (1 mentions)
Wst 1 assay, by Roche (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Wst 1 reagent, by Roche (1 mentions)
Wst 1 cell proliferation assay, by Roche (1 mentions)
Folic acid, by Merck Group (1 mentions)
8 protocols using asys uv340
1
WST-1 Cytotoxicity Assay for Chlor
2
Quantifying Intracellular Glutathione Levels
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The total glutathione (GSH) intracellular concentration in A2058, HT-1080 and SH-SY5 Y cells was obtained using the Glutathione Assay Kit (Sigma Aldrich, Milano, Italy), according to the manufacturer’s instructions. The GSH content (nmol) was normalised to cell number (1 × 106 cells) in each sample. A calibration curve was performed by using a standard curve with two-points. Briefly, GSH reacts with 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) to produce GSSG and the 1,3,5-trinitrobenzene (TNB) anion, detected by absorbance. GSSG, reduced by the enzyme glutathione reductase, releases GSH, which make a reaction with another molecule of DTNB. Furthermore, the rate of TNB production is measured compared to a single determination of how much DTNB reacts with GSH, which is proportional to the initial amount of GSH. Finally, a microplate reader Asys UV 340 (Biochrom, Cambridge, UK) was used to read the plate at 412 nm, and the GSH amount was expressed in nmol/1 × 106 cells.
3
Cell Proliferation Assay with SDT and PDT
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Effect on cell proliferation was assessed by WST-1 assay (Roche Applied Science, Penzberg, Germany) at 24, 48 and 72 h after SDT or PDT with Ala (0.45 mM in A2058 and HT-1080 cells, 1.80 mM in SH-SY5 Y cells). Briefly, after the various treatments, 1.25 × 103 A2058 and HT-1080 cells, and 2.5 × 103 SHSY5 Y cells were seeded in 100 μL of culture medium in replicates (n = 8) in 96-well culture plates (TPP). WST-1 reagent (10 μL, Sigma Aldrich) was added at 24, 48 and 72 h and the plates were incubated at 37 °C in 5% CO2 for 1.5 h for A2058 and HT-1080 cells, and for 3.5 h for SH-SY5 Y cells. The well absorbance was measured at 450 and 620 nm (reference wavelength) in a microplate reader (Asys UV340, Biochrom, Cambridge, UK).
4
Quantifying Glutathione Levels in Cell Lines
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The total glutathione level (GSSG + GSH) in HT-29, HCT116, DU154, and PC-3 cells were assayed by the Glutathione Assay Kit (Sigma, Milano, Italy), according to manufacturer’s instructions. The protein concentration (μg/mL) was quantified by the Qubit fluorometer (Invitrogen, Milan, Italy) and the Quant-IT Protein Assay Kit (Invitrogen, Milano, Italy). Calibration was performed by the application of a two-point standard curve, according to the manufacturer’s instructions.
Briefly, reduced glutathione (GSH) reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in a recycling assay and produces glutathione disulfide (GSSG) and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which release GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB react with GSH, as it is proportional to the initial amount of GSH [61 (link)]. The plate was read at 412 nm on a microplate reader Asys UV 340 (Biochrom, Cambridge, UK) and the amount of GSH was expressed in nmol/µg protein.
Briefly, reduced glutathione (GSH) reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in a recycling assay and produces glutathione disulfide (GSSG) and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which release GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB react with GSH, as it is proportional to the initial amount of GSH [61 (link)]. The plate was read at 412 nm on a microplate reader Asys UV 340 (Biochrom, Cambridge, UK) and the amount of GSH was expressed in nmol/µg protein.
5
Cytochrome c Release after SDT and PDT
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Cytochrome c was analysed at 24 h after SDT and after PDT in all cell lines, according to cell death data. Cytosolic cytochrome c was detected in supernatants from cells selectively permeabilised by an enzyme-linked immunosorbent assay (eBioscience, San Diego, CA, USA). Briefly, cells from each condition were collected by trypsin, lysed with Lysis Buffer and centrifuged and then supernatants were analysed according to the manufacturer’s instructions. The absorbance of each well was obtained at 450 nm, with a reference wavelength at 620 nm in a microplate reader (Asys UV340, Biochrom, Cambridge, UK). A standard curve based on reference standards was used to calibrate cytochrome c concentration (ng/mg of protein).
6
Quantification of Cellular Glutathione
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The total glutathione level, glutathione disulfide (GSSG) and reduced glutathione (GSH) of HT-29 and HDF 106-05 as single cell lines was determined using the Glutathione Assay Kit (Sigma-Aldrich), according to the manufacturer’s instructions. The GSH content (nmol) was normalized to protein content in each sample by quantifying cell protein concentration (μg/mL) using the Quant-iT Protein Assay Kit by using the fluorimeter Qubit (Invitrogen-Life Technologies, Milano, Italy). Calibration was performed by applying a two-point standard curve, according to the manufacturer’s instructions. Briefly, GSH reacts with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) in a recycling assay and produces GSSG and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which releases GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB reacts with GSH, as it is proportional to the initial amount of GSH [8 (link)]. The plate was read at 412 nm on a microplate reader Asys UV 340 (Biochrom, Cambridge, UK), and the amount of GSH was expressed in nmol/µg protein.
7
Squaraine Cytotoxicity Assessment in HT-1080 Cells
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C4) [15] .
5-bromine-2,3,3-trimethyl-1-butyl-3H-indolium iodide salt (3a-C4) (422.
5-iodine-2,3,3-trimethyl-3H-indole (3b) (285.1 mg) and squaric acid (57.0 mg) in butanol/toluene solution (1:1, 2 ml) at 160° C for 30 min gave 181 mg of I-NH, 0.28 mmol, 56 % yield. Sigma Aldrich) at a final concentration of 2 mM. The obtained solutions were then briefly vortexed and aliquots were stored in tightly sealed dark vials at -20°C. 1 × 10 3 HT-1080 cells were seeded in 100 µL of growth medium in replicates (n = 8) in a 96-well culture plate (TPP, Trasadingen, Switzerland). After 24 h of cell growth, the medium was removed and the cells incubated with in experimental media of differing squaraine concentrations (0.25, 0.50, 1.00 and 10 µM). The WST-1 reagent (Roche Applied Science) (10 µL/100 µL) was added, at 24, 48 and 72 h, and plates were incubated at 37°C in 5% CO 2 for 90 min. Well absorbance was measured at 450 and 620 nm (reference wavelength) in the microplate reader Asys UV340 (Biochrom, Cambridge, UK). Cell proliferation data were expressed as the ratio between the absorbance of treated and untreated cells.
5-bromine-2,3,3-trimethyl-1-butyl-3H-indolium iodide salt (3a-C4) (422.
5-iodine-2,3,3-trimethyl-3H-indole (3b) (285.1 mg) and squaric acid (57.0 mg) in butanol/toluene solution (1:1, 2 ml) at 160° C for 30 min gave 181 mg of I-NH, 0.28 mmol, 56 % yield. Sigma Aldrich) at a final concentration of 2 mM. The obtained solutions were then briefly vortexed and aliquots were stored in tightly sealed dark vials at -20°C. 1 × 10 3 HT-1080 cells were seeded in 100 µL of growth medium in replicates (n = 8) in a 96-well culture plate (TPP, Trasadingen, Switzerland). After 24 h of cell growth, the medium was removed and the cells incubated with in experimental media of differing squaraine concentrations (0.25, 0.50, 1.00 and 10 µM). The WST-1 reagent (Roche Applied Science) (10 µL/100 µL) was added, at 24, 48 and 72 h, and plates were incubated at 37°C in 5% CO 2 for 90 min. Well absorbance was measured at 450 and 620 nm (reference wavelength) in the microplate reader Asys UV340 (Biochrom, Cambridge, UK). Cell proliferation data were expressed as the ratio between the absorbance of treated and untreated cells.
8
Cell Proliferation Assay for Targeted Therapeutics
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The WST-1 cell proliferation assay (Roche Applied Science, Penzberg, Germany) was used to evaluate the effects of treatment on cell growth. After the various treatments, 2.5 × 10 3 MCF7, 1.5 × 10 3 HCT-116 and 2.0 × 10 3 KB cells were seeded in 100 μl of culture medium in replicates (n = 8) for each condition in 96-well culture plates (TPP, Trasadingen, Switzerland). MCF7 and HCT-116 cells were incubated for 1.5 h and KB cells for 2 h with WST-1 reagent (10 μl) at 37°C, 24, 48 and 72 h after the US treatment. Well absorbance was measured at 450 and 620 nm (reference wavelength) on a microplate reader (Asys UV340; Biochrom, Cambridge, UK). Cell proliferation data were expressed as a percentage of untreated cells.
Folic acid uptake competition assay HCT-116 and KB cells were also incubated with FFD-MEM medium containing 1.0 nM FA-PEG-GNP suspension and 200 μM Folic acid (Sigma) for 2 h to evaluate the uptake selectivity of FA-PEG-GNP by cell folate receptor under competition conditions with Folic acid. Cells were then detached and subjected to US treatment as previously described; cell growth was evaluated using a WST-1 assay after 24, 48 and 72 h.
Folic acid uptake competition assay HCT-116 and KB cells were also incubated with FFD-MEM medium containing 1.0 nM FA-PEG-GNP suspension and 200 μM Folic acid (Sigma) for 2 h to evaluate the uptake selectivity of FA-PEG-GNP by cell folate receptor under competition conditions with Folic acid. Cells were then detached and subjected to US treatment as previously described; cell growth was evaluated using a WST-1 assay after 24, 48 and 72 h.
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