CCK8 assay: Cells were plated in 96-well plates (2000 cells/well in 100 µL), cultured overnight, and incubated with 0, 0.99, 2.96, 8.89, 26.67 and 80 μg mL−1 NPs for 24 h. Then, 10 µL of cell counting kit-8 (CCK-8, Dojindo, Japan) was added, and the cells were incubated at 37 °C in the dark for 4 h. The optical density at 450 nm was determined with a microplate reader. Live/dead, EdU, and ROS measurements: OCM1a cells were pretreated for 24 h with PBS, 15 µg mL−1 ICG-MOF-PR, ICG-OP@MOF-PR, ICG-Cis@MOF-PR and ICG-COP@MOF-PR. Cell survival (calcein AM staining) and death (PI staining) were assessed using a live/dead kit (Beyotime, China) according to recommended procedures. Cell proliferation was studied using a Cell-Light EdU Apollo488 In Vitro Kit (RiboBio, China) following the manufacturer’s instructions. The ROS levels were assessed using a DCFH-DA assay (Beyotime, China) according to recommended procedures. Images were taken with a fluorescence microscope (Nikon).
Dcfh da assay
Manufactured by Beyotime
Sourced in China
DCFH-DA assay is a fluorometric method used to measure the levels of reactive oxygen species (ROS) in cells. It utilizes the cell-permeable compound 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) which is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) upon oxidation by ROS.
Automatically generated - may contain errors
Lab products found in correlation
Cell light edu apollo488 in vitro kit, by RiboBio (1 mentions)
Live dead kit, by Beyotime (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Hoechst 33342 solution, by Beyotime (1 mentions)
Cellquest software version 5, by BD (1 mentions)
Cck 8 assay, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Software version 10, by FlowJo (1 mentions)
Flow cytometry, by BD (1 mentions)
8 protocols using dcfh da assay
1
CCK-8 Assay and Cell Viability Evaluation
2
ROS Detection in PDLSCs
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DCFH-DA assay (Beyotime, Shanghai, China) was used to detect the levels of ROS in PDLSCs. After plating the cells onto 24-well culture dishes (2 × 104/well) overnight, the cells were treated with tFNAs or LPS for 6 h, followed by incubation with a DCFH-DA probe for 20 min. To locate the cell nucleus, Hoechst 33342 solution (Beyotime, Shanghai, China) was used to stain the live cells. Finally, the sample images were captured using an inverted fluorescence microscope (DMi8, Leica, Wetzlar, Germany).
3
Measuring Oxidative Stress Levels via DCFH-DA
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ROS were detected in different groups (Control, H2O2, LBP1 + H2O2, LBP2 + H2O2, LBP3 + H2O2) with a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay (Beyotime Institute of Biotechnology). After 6 h of 250 µmol/l H2O2 treatment, cells, treated with different concentrations (100, 200 and 400 µg/ml) of LBP for 6 h, were seeded into wells of 6-well plate. DCFH-DA (10 µmol/l) was subsequently added into each well. After incubation for 20 min at 37°C, cells were rinsed with PBS and analyzed by flow cytometry. ROS levels were analyzed by CellQuest software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA) and results were calculated relative to the control group.
4
ROS and MMP Modulation in C. albicans
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ROS were detected by the DCFH-DA assay (Beyotime Biotech, Shanghai, China). MMP was detected by the Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime Biotech, Shanghai, China). The C. albicans cells (1 × 107 CFU/mL) were cultured with various concentrations of TE1 and ASA (0, 1/4 MIC, 1/2 MIC, and 1 MIC) for 2 h at 30 °C. Then, the treated cells were washed twice with PBS and stained with DCFH-DA or JC-1 for 30 min in the dark. Subsequently, the cells were washed with PBS and resuspended in PBS post-centrifugation. Finally, the absorbance was measured using a Synergy H1 microplate reader. CCK-8 assay (Beyotime Biotech, Shanghai, China) was used to measure viable cell numbers. The percentage of control was used to express ROS. The MMP was determined as the ratio of JC-1 monomer (490/530 nm) and JC-1 aggregate (525/590 nm) fluorescence intensity. All experiments were performed in triplicate.
5
Measuring Oxidative Stress with DCFH-DA
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The treated cells were washed with PBS twice, and then tested by a 2,7-dichlorofluorescein diacetate (DCFH-DA) assay (Beyotime, China), set up a positive control group (Rosoup reagent) before adding DCFH-DA and incubate at 37°C for 20 minutes. After discarding the supernatant and washing twice with PBS, the cells were incubated with DCFH-DA (10 μmol/L) at 37°C for 30 minutes. The fluorescence intensity was read by a plate reader (Protein Simple, USA) (excitation wavelength of 488 nm and emission wavelength of 525 nm).
6
ROS Detection in IL-1β-Treated Cells
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Reactive oxygen species (ROS) were detected in different groups (control, rh-IL1B) using a 2,7‑dichlorofluorescin diacetate (DCFH‑DA) assay (Beyotime Institute of Biotechnology, Haimen, China). After 48 h of treatment with rh-IL1B (50 ng/mL, 201-LB-025, R&D Systems, USA), DCFH‑DA (10 µmol/L) was added to each well. After incubation for 20 min at 37 °C, the cells were rinsed with PBS and analyzed by flow cytometry. ROS levels were analyzed using FlowJo software (version 10.07 (FlowJo LLC, USA), and the results were calculated relative to the control group.
7
Quantifying Myocardial ROS Levels
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The level of ROS in myocardial cells was determined using the DCFH-DA assay strictly according to the instructions of kit (Beyotime Biotechnology): after the experiment, the rats were sacrificed immediately, the heart was taken, and the left ventricle was separated. Then, the tissues were homogenized with an appropriate amount of normal saline and centrifuged at 10,000 g and 4°C for 15 min. An appropriate amount of supernatant was taken, added with 10 μM of DCFH-DA, incubated at 37°C for 20 min in the dark, and washed with PBS and washing solution, followed by photography under a laser confocal microscope. Finally, the content of ROS in myocardial cells was calculated.
8
Quantifying Intracellular Hydroxide Levels
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Intracellular OH− levels were detected using a DCFH-DA assay (Beyotime Institute of Biotechnology, Jiangsu, China). All cells were pretreated with z-VAD-fmk. Briefly, following 48 h of treatment in the different groups, the cells were harvested at a concentration of 2×106 cells/ml and labeled with DCFH-DA (10 µM) in a humid atmosphere of 5% CO2, shielded from light, for 20 min. Subsequently, all labeled cells were washed and collected. Flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) was performed in order to detect the fluorescence intensity. The OH− level was determined as the percentage of labeled (DCFH-DA) to gated cells.
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