DPPP Method for Determining Lipid Peroxidation Levels in Live Cells[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Dissolve DPPP (Diphenyl-1-pyrenylphosphine) in DMSO to prepare a stock solution. Store at -20°C under dark conditions with nitrogen protection. Dilute with PBS to the working concentration (e.g., 167µM) before use. Prepare complete cell culture medium (containing 10% fetal bovine serum, 50µg/ml penicillin, and 50µg/ml streptomycin).
2. Cell Culture and DPPP Labeling: Culture the target cells routinely at 37°C with 5% CO₂. For the experiment, collect the cells and wash three times with PBS. Resuspend in PBS (density approximately 1×10⁸ cells/ml) and pre-incubate at 37°C for 5 minutes. Add the DPPP working solution (final concentration 167µM) and incubate at 37°C in the dark for 5 minutes. After incubation, wash the cells three times with PBS to remove unincorporated DPPP. Finally, resuspend in an appropriate buffer or culture medium for subsequent use. All operations must be performed in light-shielded containers.
3. Lipid Peroxidation Induction and Treatment: Suspend the DPPP-labeled cells (density approximately 3×10⁶ cells/ml) in growth medium. Add the required inducer (e.g., DEM, AAPH, etc.) or replace with medium containing different serum concentrations (e.g., serum-free medium). Incubate at 37°C in a CO₂ incubator in the dark for the specified duration. A vitamin E (α-tocopherol) treatment group can be set up as an antioxidant control.
4. Fluorescence Detection: Use a fluorescence spectrophotometer set to an excitation wavelength of 351nm and an emission wavelength of 380nm to measure the sample fluorescence intensity. Alternatively, use a fluorescence microscope to directly observe the fluorescence distribution of DPPP oxide in the cells (staining in the cytoplasmic membrane region, no staining in the nucleus).
Precautions:
(1) DPPP has no significant effect on cell proliferation, viability, or morphology and can be used for long-term peroxidation experiments for at least 3 days.
(2) DPPP oxide is very stable within the membranes of live cells, with no significant change in fluorescence signal for at least 2 days.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Takahashi M, Shibata M, Niki E. Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine. Free Radic Biol Med. 2001 Jul 15;31(2):164-74. |
