The levels of intracellular ROS were measured using DCFH-DA fluorescent dye (Beyotime, Nanjing, China) according to the manufacturers′ recommendations. Briefly, cells were cultured in six-well tissue culture plates and incubated with indicated concentrations of SFN for 24 h. As a positive control, the cells were exposed to pyocyanin (an ROS inducer, 200 µM) for 3 h. After treatment, cells were incubated with 10 µM DCFH-DA diluted in serum-free culture medium for 30 min at 37 °C in the dark. After incubation, the cells were washed twice with PBS and micrographs obtained using a conventional fluorescent microscope (Olympus).
Conventional fluorescent microscope
Manufactured by Olympus
Sourced in Japan
A conventional fluorescent microscope is an optical microscope that uses fluorescence to visualize specimens. It illuminates the sample with a specific wavelength of light, causing fluorescent molecules within the sample to emit light at a longer wavelength, which is then detected and displayed as an image.
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9 protocols using conventional fluorescent microscope
1
Intracellular ROS Measurement via DCFH-DA
2
Dihydroethidium Fluorescence Assay
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Cells were incubated with 10 μM of DHE at 37°C for 30 min, washed twice with PBS, and microphotographed under a conventional fluorescent microscope (Olympus, Japan) immediately. For each well, 5 fields were taken randomly. Then, cells were rapidly digested, harvested and washed twice with cold PBS, and detected by FCM. The DHE Fluorescence intensity was measured and quantified at an excitation wave length of 518 nm through PE filters (23 (link), 24 (link)).
3
Quantifying Intracellular ROS and GSH
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Intracellular ROS level was measured using DCFH-DA as reported previously [9 (link), 10 (link)]. Briefly, PL-treated cells were incubated with 10 μM of DCFH-DA for 30 min at 37°C, washed twice with PBS and then micrographed with a conventional fluorescent microscope (Olympus, Tokyo, Japan). For each culture, a minimum of 9 random fields were captured. Average fluorescent intensity was analyzed using the Image-Pro Plus software (Media Cybernetics, USA). For GSH measurement, PL-treated HepG2 or Huh7 cells were washed with ice-cold PBS three times, scrapped off from the plates and subjected to sonification. The supernatants were collected and reduced GSH was calculated according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute).
4
Intracellular ROS Measurement Assay
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Intracellular ROS levels were measured using 2′,7′-dichlo-rodihydrofluorescein diacetate (DCFH-DA; Beyotime, Nanjing, China), according to the manufacturer’s recommendations. Briefly, HeLa cells in six-well tissue culture plates were treated with dicerandrol B (3 or 5 µg/mL) or DMSO (untreated control) in DMEM for 24 hours; some cells were pretreated with 100 µmol/L N-acetyl-l -cysteine (NAC; antioxidant) for 60 minutes. After treatment, cells were incubated with 10 µM DCFH-DA diluted in serum-free culture medium in the dark at 37°C for 30 minutes, washed twice with PBS, and micrographs were obtained using a conventional fluorescent microscope (Olympus).
5
Measuring Intracellular Superoxide Levels
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Cells were incubated with 10 μM of DHE for 30 min at 37°C, washed twice with PBS, and immediately microphotographed under a conventional fluorescent microscope (Olympus, Japan). For each well, 5 fields were taken randomly.
6
Protein Detection and Localization
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Western blot analysis and fluorescent cytoimmunostaining were performed as described previously [45 (link)]. Briefly, equal amounts of total proteins were subjected to mini-PAGE gel electrophoresis and transferred to NC membranes. The blots were incubated with corresponding primary and fluorescent secondary antibodies. The bands were quantified using the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NE, USA). β-actin was used as internal control. For fluorescent cytoimmunostaining, PL-treated cells in the 35-mm culture dishes were fixed, permeabilized, blocked, and then incubated with corresponding primary and Dylight 488-labeled secondary antibodies (Abbkine, Redlands, CA, USA). Micrographs were taken under the same conditions with a conventional fluorescent microscope (Olympus, Tokyo, Japan).
7
Intracellular ROS Detection by DCFH-DA Staining
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Intracellular ROS production was determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining followed by fluorescent microscope [22 (link)]. Briefly, cells were incubated with 10 μM DCFH-DA solution at 37°C for 0.5 h, washed with PBS twice, and photographed under a conventional fluorescent microscope (Olympus, Tokyo, Japan). For each culture, a minimum of 9 random fields were captured.
8
Intracellular Calcium Imaging with Fluo-3 AM
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Intracellular Ca2+ levels were detected with Fluo-3 AM as reported [46 (link)]. PL-treated cells were washed with Ca2+-free PBS three times and then incubated with 5 μM of Fluo-3AM for 30 min. After brief rinse with Ca2+-free PBS, living cells were micrographed immediately under a conventional fluorescent microscope (Olympus, Tokyo, Japan).
9
Immunofluorescence Staining Protocol for Cells and Tissues
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Immunofluorescence staining was performed as previously reported (29 (link)). Cells in 35-mm culture dishes were fixed, permeabilized, blocked, and then incubated with primary and corresponding Dylight 488-labeled secondary antibodies (Abbkine, CA, USA). F-actin was stained with rhodamine-phalloidin (Yisheng Bioengineering Institute, China) according to the manufacturer's instructions. Hoechst 33342 was used to stain the nucleus. For paraffin-embedded tissues, rat brain slices were deparaffinized, rehydrated, antigen unmasked, blocked with 5% bovine serum albumin (BSA) and then incubated with primary antibodies and corresponding Dylight 488/594- labeled secondary antibodies. Micrographs were taken under the same conditions with a conventional fluorescent microscope (Olympus, Tokyo, Japan).
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