Spectramax i3x spectrophotometer

The intracellular concentrations of reduced (L-γ-glutamyl-L-cysteinylglycine), oxidized (GSSG; glutathione disulfide), and total glutathione were evaluated using a GSH, GSSG, and total glutathione determination kit (Abcam, Cambridge, UK). Cells treated with 200 μg/mL XOS and controls grown in a complete medium without XOS for 24 h were used in the experiment. To determine GSH, GSSG, and total glutathione concentrations, the protocol supplied by the manufacturer of the kit was strictly followed. Results were obtained by fluorescent intensity quantification of the samples using a SpectraMax i3x spectrophotometer (Molecular devices, San Jose, CA, USA) at Excitation/Emission ¼ 350/470 nm. All samples were assayed in triplicates.

SH-SY5Y human neuroblastoma cells are related to aging, and the viability of SH-SY5Y cells were detected by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide) assay [31 (link)]. Cells were seeded overnight at a density of 10⁵ cells/well 96-well plates in 100 μL medium. The Cells were co-incubated with 20 μM CP-6 with streptozotocin (STZ, 0.8 μM) for 24 h. Then, the medium was removed and added 0.5 mg/μL MTT. After incubation at 37 °C for 4 h, 100 μL of dimethyl sulfoxide (DMSO) was added to SH-SY5Y cells each well, and the mixture was shaken at a low speed for 15 min to fully dissolve the formazan crystals, followed by the measurement of the absorbance at 490 nm using an SpectraMax i3x spectrophotometer (Molecular Devices, USA).

EPS matrix detection was performed as described previously 72. In brief, 130 l diluted bacterial cultures (1:50 from an overnight bacterial) in LB, LB + 0.15% bile or LB + 0.2% NaGCH were grown in a black 96-well clear flat bottom polystyrene plate (Corning® Cat. No. CLS3603 Sigma) overnight at 37 °C. Planktonic bacteria was transferred to a new 96-well polystyrene microtiter plate (Corning®, Cat. No. CLS3595 Sigma). Wells in the black plate were fixated with formaldehyde/glutaraldehyde, rinsed with PBS and stained with 150 l of Concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC) (25 g/ml) for 15 min. After rinsing with PBS, the amount of retained ConA-FITC was measured using a SpectraMax i3x spectrophotometer (Molecular Devices) at 480 nm. This assay was performed in four independent experiments.

Biofilm assays were performed on microtiter plates as described previously (61 (link)). Overnight cultures were diluted 1:100 in fresh LB medium without salt. Aliquots (150 μl) of the diluted samples were incubated in the wells of a flat-bottomed 96-well polystyrene microtiter plate (Corning) for 48 h at 28 or 37°C. Each cell culture (100 μl) was transferred into another flat-bottom 96-well polystyrene microtiter plate and spectrophotometrically measured at 600 nm to assess planktonic bacterial growth. After the planktonic bacteria were discarded from the initial microtiter plate, bacterial cells bound to the wells were gently washed twice with 200 μl of phosphate-buffered saline (PBS), air dried, and then stained with 200 μl of 0.1% (wt/vol) crystal violet for 15 min. After incubation, the stained plates were washed four times with distilled water to remove excess crystal violet, and the stained biofilms were solubilized with 200 μl of 80% ethanol. The absorbance (540 nm) was measured with a SpectraMax i3x spectrophotometer (Molecular Devices). For each experiment, the absorbance of the blank control with crystal violet was subtracted from the experimental sample. Three replicates were used in each experiment, and three independent experiments were performed. Biofilm production was classified as low, medium, or high as previously described (62 (link)).