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Principle, Application Method, and Precautions for Lipid Peroxidation Sensors
Lipid peroxidation is the oxidative degradation of lipids, primarily initiated by reactive oxygen species (ROS). The Lipid Peroxidation Sensor detects lipid peroxidation in living cells through the oxidation of a C11‑BODIPY 581/591 probe. C11 BODIPY 581/591 is essentially a lipophilic fluorescent dye that exhibits excellent photostability and minimal fluorescence artifacts, allowing it to accumulate within cellular membranes. When the polyunsaturated butadiene moiety of the dye undergoes oxidation, the peak wavelength of its maximal fluorescence emission shifts from approximately 590 nm to about 510 nm, causing the fluorescence of live cells to change from red to green while retaining its lipophilicity, thereby reflecting the level of lipid peroxidation in the membrane. This sensor is commonly employed in fluorescence microscopy and flow cytometry to visualize and monitor the generation and dynamic changes of lipid‑derived ROS.
Principle of measurement
LPO is heated under acidic conditions, yielding the small-molecule end product MDA. MDA condenses with thiobarbituric acid (TBA) to form a red-colored product that exhibits a maximum absorption peak at 535 nm.
Instructions for Use
1. Flow cytometry:
(1) Suspension cells: Centrifuge at 800 g for 5 min to pellet the cells; then wash the cells twice with PBS.
(2) Adherent cells: Discard the culture medium, gently wash the cells twice with room‑temperature PBS or culture medium, then digest with trypsin. Centrifuge at 800g for 5 minutes to pellet the cells, followed by two washes with PBS. Collect 1–5 × 10^5 cells per sample. [Note]: Handle the cells gently during pipetting; avoid prolonged digestion, as vigorous pipetting or over‑digestion with trypsin may compromise the experimental results.
(3) The recommended final concentration of the lipid peroxidation sensor for detecting lipid peroxidation is 5 μM. Preparation: Prepare a staining solution of the lipid peroxidation sensor at a final concentration of 5 μM using fresh, serum-free culture medium; the DMSO content should be ≤0.5%. [Note]: It is advisable to complete this step prior to cell digestion to prevent prolonged incubation after digestion from compromising experimental results.
(4) Add the pre‑prepared lipid peroxidation staining solution to the collected cell pellet, using a recommended volume of 500 μL. Incubate at 37°C for 30 minutes, gently vortexing the sample every 5–10 minutes to resuspend the cells and ensure thorough staining.
(5) After staining, centrifuge at 800 g for 5 min, discard the supernatant, then wash the cells twice with PBS. Resuspend the cells in 200 μL of PBS for flow cytometric analysis.
2. In situ imaging:
(1) Seed cells at an appropriate density onto 20-mm coverslips (or use a confocal dish as an alternative), and treat them with the experimental drug for an appropriate duration.
(1) After the incubation period, aspirate the culture medium and wash the cells once with PBS; add 500 μL of the prepared lipid peroxidation staining solution and stain at 37°C for 30 minutes; refer to the staining solution preparation protocol used in flow cytometry for the preparation method.
(2) Discard the culture medium, wash the cells twice with PBS, and then perform imaging analysis under a fluorescence microscope or a laser confocal microscope. Cells should be imaged within 1 hour after staining.
Complete the testing within (2).
Test conditions
In its reduced state, this molecular probe exhibits an optimal excitation wavelength of 581 nm and an emission wavelength of 591 nm; upon oxidation by lipid‑derived ROS, the optimal excitation wavelength shifts to 500 nm, with emission at 510 nm. Consequently, in flow cytometry, the detection channel is FL1‑A; for confocal microscopy, the oxidized form can be excited at 488 nm (FITC channel), while the reduced form can be excited at 561 nm (TRITC channel).
Precautions
1. Before use, ensure that Reagent II is completely dissolved; if it has not dissolved, heat it to 70–90°C and shake vigorously to facilitate dissolution.
2. Before use, centrifuge carefully for a few seconds to dispense the product; wear gloves during handling and avoid contact with skin, eyes, and mucous membranes.
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