DMA-SiRd

Operating Instructions (The following is a recommended experimental protocol for guidance only, and should be adjusted according to your specific requirements)
1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: Prepare an appropriate concentration of DMA-SiRd stock solution using DMSO, and store it at -20°C in the dark.
2. Working Solution Preparation
2.1 Diluent: No specific diluent is specified for cell staining; the stock solution can be directly injected for in vivo experiments.
2.2 Working concentrations: 5 μM, 3 mg/mL, 12.5 mg/mL.
2.3 Notes: The concentration of the working solution can be adjusted as needed; prepare it fresh before use.
3. Staining Procedures
3.1 Adherent cells^[1]:
3.1.1 Sample type description: HeLa cells and HepG2 cells; trypsinization is not required for imaging experiments.
3.1.2 Incubation conditions: Incubate with 5 μM DMA-SiRd for 30 min.
3.2 Tissue sections^[1]:
3.2.1 Sample type description: Frozen sections of mouse thoracic aorta, mouse brain tissue, blood vessels.
3.2.2 Incubation conditions: Inject DMA-SiRd via tail vein (3 mg/mL, 125 μL per mouse or 12.5 mg/kg), and collect samples 20 min after injection.
4. Detection and Analysis
4.1 Instrument types: Fluorescence lifetime imaging microscope (FLIM), two-photon fluorescence lifetime imaging microscope (TP-FLIM), confocal fluorescence microscope, super-resolution fluorescence microscope.
4.2 Excitation/emission wavelengths:
4.2.1 Single-photon excitation/emission: Red channel (mitochondria): λ_ex=633 nm, λ_em=650-750 nm; Green channel (lipid droplets): λ_ex=546 nm, λ_em=570-620 nm.
4.2.2 Two-photon excitation: 1040 nm (lipid droplets), 1200 nm (mitochondria/blood vessels).
4.3 Result analysis:
4.3.1 Fluorescence localization: The red channel signal localizes to mitochondrial cristae; the green channel signal localizes to lipid droplets; the above signals can be used to visualize lipid droplet-mitochondria contact sites.
4.3.2 Changes in fluorescence lifetime and intensity correlate with changes in local polarity; prolonged lifetime indicates increased content of low-polarity lipids, while shortened lifetime indicates decreased content of low-polarity lipids.
4.3.3 Heterogeneity detection: Atherosclerotic plaques exhibit heterogeneous lifetime distribution; exposure to ferroptosis inhibitors restores lifetime uniformity.
4.3.4 In vivo brain imaging: The lifetime of vascular lipid plaques in atherosclerotic mice is longer than that of lipids in brain parenchyma.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.