GSH and GSSG Assay Kits (Beyotime, S0053, Shanghai, China) were used to measure the content of GSH, and then they were standardized to tissue weight. With the indicated treatments, the LPO MDA Assay Kit (Beyotime, S0131S, Shanghai, China) was used to determine the MDA contents. Briefly, the samples were centrifuged after being treated with lysis buffer, then supernatants were mixed with TBA detection solution to measure the absorbance at 532 nm, and then the contents of MDA were calculated according to the standard curve. Finally, the data were standardized to tissue weight.
Lpo mda assay kit
Manufactured by Beyotime
Sourced in China
The LPO MDA assay kit is a laboratory reagent used to measure the level of malondialdehyde (MDA), a biomarker of lipid peroxidation, in biological samples. The kit provides the necessary reagents and protocols to quantify MDA levels colorimetrically.
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4 protocols using lpo mda assay kit
1
Quantifying Oxidative Stress Markers
2
Quantifying Lipid Peroxidation Markers
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MDA levels in cells were measured using an LPO MDA assay kit (Beyotime Biotechnology). Cellular proteins were extracted and added to MDA detection working solution and then heated in boiling water for 15 min. The absorbance was measured at 532 nm using a microplate reader to calculate the MDA levels. 4-HNE levels were determined using a 4-HNE assay kit (Abcam Inc., Cambridge, MA, USA) according to the manufacturer’s instructions, and the OD values of the cells were determined using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at 450 nm. Then, a standard curve was drawn based on the OD values of the standard, and the concentration of the samples was calculated.
3
Oxidative Stress and Antioxidant Assays
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ROS were detected using reactive oxygen species assay kit (Beyotime, China). The content of MDA was monitored using LPO MDA assay kit (Beyotime, China). The GSH detection kit (Solarbio Science, China) was used to detect GSH in CRC cells.
4
Intracellular Oxidative Stress Assessment
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Cells were cultured in 6-well plates with a density of 5×105 cells per well and incubated for 12 hours. After applying various treatments, the cells underwent the following procedure to assess intracellular oxidation levels.
For intracellular GSH detection, MNNG/HOS cells were suspended in 200 µL of PBS per well and disrupted using ultrasonication. The disrupted cells were mixed with GSH probe precipitant in a 1:1 ratio (v/v) and then centrifuged at 3500 rpm for 10 minutes. The supernatant’s absorbance at 405 nm was measured, and GSH levels were determined using a GSH assay kit as per its instructions. Intracellular lipid peroxidation (LPO) was assessed by measuring MDA levels using an LPO MDA assay kit (S0131S, Beyotime, Shanghai, China) following established methods.32 (link) For visualizing cellular ROS, the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed. DCFH-DA emits green fluorescence upon reacting with ROS. The treated cells were rinsed, stained with DCFH-DA (10 µM) for 20 minutes, and then washed with PBS twice. Subsequently, the cells were observed and imaged using a fluorescence microscope.
For intracellular GSH detection, MNNG/HOS cells were suspended in 200 µL of PBS per well and disrupted using ultrasonication. The disrupted cells were mixed with GSH probe precipitant in a 1:1 ratio (v/v) and then centrifuged at 3500 rpm for 10 minutes. The supernatant’s absorbance at 405 nm was measured, and GSH levels were determined using a GSH assay kit as per its instructions. Intracellular lipid peroxidation (LPO) was assessed by measuring MDA levels using an LPO MDA assay kit (S0131S, Beyotime, Shanghai, China) following established methods.32 (link) For visualizing cellular ROS, the fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was employed. DCFH-DA emits green fluorescence upon reacting with ROS. The treated cells were rinsed, stained with DCFH-DA (10 µM) for 20 minutes, and then washed with PBS twice. Subsequently, the cells were observed and imaged using a fluorescence microscope.
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