S (v/v). Cells had been maintained at 37 C inside a saturated humidity atmosphere containing 5 CO2 . 2.four. Cell Viability Assay HT22 cells (5 103 cells/well) were seeded and cultured into a 96-well plate for 24 h. The cells were pretreated with CIE at concentrations of 50, one hundred, or 200 /mL for 2 h and with H2 O2 (500) for 24 h. In total, 10 of CCK solutions was added to every nicely, and also the cells have been incubated for yet another 1 h. The absorbance was evaluated applying a microplate reader (SpectraMax i3, Molecular Devices, San Jose, CA, USA) at 450 nm. two.five. Lactate Dehydrogenase Assay The release of lactate dehydrogenase (LDH) within the medium was evaluated working with the LDH assay kit, in accordance with the manufacturer’s instruction. Right after therapy with 500 H2 O2 for 24 h, the cells have been centrifuged, and ten culture medium was transferred to a 96-well plate. The reaction remedy (10 /well) was applied to every single effectively, plus the LDH activity was investigated utilizing a microplate reader at 450 nm. The LDH release 16 Prostaglandin Receptor outcomes were expressed as percentage in the control. 2.six. Intracellular ROS Determination Intracellular ROS was detected with an ROS detection kit, in accordance with the manufacturer’s instruction. Right after CIE and H2 O2 remedies, the cells have been washed twice in 1assay buffer and had been then incubated with 20 of H2 DCFDA for 30 min at 37 C in the dark. The distribution of DCF fluorescence was analyzed employing a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The representative fluorescence photos were obtained with a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, Japan). 2.7. Flow Cytometry Analysis The presence of apoptotic cells was determined employing the Annexin V-FITC/PI apoptosis detection kit. The cells were harvested 24 h after exposure to H2 O2 , washed with ice-cold Ca2 -free phosphate-buffered saline, and resuspended in binding buffer (0.1 M HEPES/NaOH, pH 7.4, 1.4 M NaCl, and 25 mM CaCl2). Next, five of Annexin V-FITC and 5 of PI had been added and incubated for 15 min at area temperature (RT) in the dark. The cells were analyzed by means of flow cytometry (FACSCalibur, Becton Dickinson, CA, USA). two.eight. JC-1 Staining JC-1, a fluorescent probe, was applied to detect the mitochondrial membrane prospective, as outlined by the manufacturer’s instruction. Briefly, JC-1 operating remedy (five mg/mL) was added to the medium for 15 min at 37 C within the dark and was rinsed with culture media. Fluorescence pictures have been obtained working with a fluorescence microscope. The red (polymeric) fluorescence was visualized beneath an excitation wavelength of 520 nm and an emission wavelength of 590 nm. The green (monomeric) fluorescence was visualized below an excitation wavelength of 490 nm and an emission wavelength of 530 nm. 2.9. Western Blot Analysis For Western blot analysis, the total cellular proteins have been lysed employing radioimmunoprecipitation assay lysis buffer (Millipore) by adding protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration was determined employing the Bradford method. Equivalent amounts of total protein have been separated by means of sodium dodecyl sulfate olyacrylamide gel electrophoresis and were electrically transferred onto the PVDF membranes. The membranes had been blocked with three BSA at RT for 1 h and had been incubated with their respective main QX-314 Cancer antibodies (1:1000) overnight at four C. Following washing, theyNutrients 2021, 13,4 ofwere incubated with HRP-conjugated secondary antibodies for 1 h at RT. The relative i.
