Lipid peroxidation is the process of oxidative degradation of lipids, mainly triggered by reactive oxygen species (ROS). The lipid peroxidation sensor detects lipid peroxidation in living cells through the oxidation of a C11-BODIPY 581/591 reagent. C11 BODIPY 581/591 is essentially a lipophilic fluorescent dye with good photostability and low fluorescence artifacts, which can accumulate in the membrane. Once the polyunsaturated diene group of the dye is oxidized, the maximum emission wavelength peak of fluorescence shifts from about to 590 nm to about to 510 nm, and the fluorescence of living cells changes from red to green, but still maintains lipophilicity, reflecting the level of lipid peroxidation in the membrane. This type of sensor is commonly used in fluorescence microscopy and flow cytometry analysis to visually monitor the generation and dynamic changes of lipid ROS.
Measurement principle
LPO is heated under acidic conditions to produce small molecule end product MDA, which condenses with thiobarbituric acid (TBA) to form a red product with a maximum absorption peak at 535nm.
usage method
1. Flow cytometry analysis:
(1) Suspension cells: Centrifuge at 800 g for 5 minutes to collect cell pellet; Then wash the cells twice with PBS.
(2) Adhesive cells: Discard the culture medium and wash the cells lightly twice with room temperature PBS or culture medium. Then digest with trypsin and centrifuge at 800g for 5 minutes to collect cell precipitates. Wash the cells twice with PBS, and collect 1-5 × 105 cells from each sample. [Note]: During the operation, gently blow and beat the cells, and the digestion time should not be too long. Intense blowing or excessive digestion by pancreatic enzymes may affect the experimental results.
(3) The recommended concentration for lipid peroxidation detection using a lipid peroxidation sensor is a final concentration of 5 μ M; Preparation method: Prepare a lipid peroxidation sensor staining solution with a final concentration of 5 μ M using fresh blank culture medium for later use, with DMSO content ≤ 5 ‰. [Note]: It is recommended to prepare the cells before digestion to avoid prolonged storage after digestion, which may affect the experimental results.
(4) Add the pre prepared lipid peroxidation staining solution to the collected cell pellet, with a recommended volume of 500 μ L. Then place it in a 37 ℃ incubator and stain for 30 minutes. During this period, lightly tap the cells at 5-10 minute intervals to suspend them and ensure more thorough staining.
(5) After staining, centrifuge at 800 g for 5 minutes, discard the supernatant, and then wash the cells twice with PBS. Add 200 μ L PBS to resuspend the cells for flow cytometry analysis.
2. In situ imaging:
(1) Inoculate cells of appropriate density onto a 20mm cover glass (confocal dish can also be used as a substitute), and administer experimental drug treatment appropriately
(1) Afterwards, aspirate the culture medium and wash the cells once with PBS; Add 500 μ L of prepared lipid peroxidation staining solution and stain at 37 ℃ for 30 minutes; The preparation method refers to the preparation of staining solution in flow cytometry analysis.
(2) Discard the culture medium, wash the cells twice with PBS, and perform imaging detection under fluorescence microscopy or laser confocal microscopy. Cells should be stained within 1 hour after staining
(2) Complete the testing internally.
Testing conditions
The optimal excitation wavelength and emission of the molecular probe in the reduced state are 581 nm and 591 nm, respectively; The optimal excitation wavelength after oxidation by Lipid ROS is 500 nm, with emission at 510 nm. Therefore, in flow analysis, the detection channel is FL1-A; Laser confocal detection: The oxidation state excitation wavelength can be selected as 488nm (FITC) channel; The excitation wavelength of the reduced state can be selected as 561 nm (TRITC) channel.
matters needing attention
1. Before use, pay attention to whether reagent 2 is completely dissolved. If it is not dissolved, it can be heated at 70 ℃ -90 ℃ and shaken to promote dissolution.
2. Be careful to centrifuge for a few seconds before use. Wear gloves and avoid contact with skin, eyes, and mucous membranes.
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