CQPP

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: 1,4-dioxane, DMSO, or Tris-HCl buffer.
1.2 Concentration recommendation: 2 μM, 5 μM, or 2 mM.
Note: Paper ^[1] uses stock concentrations of 2 μM or 5 μM (used directly without dilution), while Paper ^[2] uses a 2 mM stock concentration (diluted to working solution).

2. Working Solution Preparation
2.1 Diluent: Cell culture medium.
2.2 Working concentration: 5 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For adherent cells (HT-1080 cells)^[1][2]:
3.1.1 Incubation conditions:
3.1.1.1 Incubate with 5 μM CQPP for 2 h.
3.1.1.2 Co-incubate with 5 μM CQPP and 2 μM ferroptosis inducer for up to 4 h.
3.1.1.3 For pre-treated samples, incubate with 10 μM ferroptosis inducer for 6 h prior to CQPP staining.
3.1.2 Washing steps: No washing steps required.

4. Controls
4.1 Negative control: Untreated HT-1080 cells.
4.2 Apoptosis control: HT-1080 cells treated with cisplatin.

5. Detection & Analysis
5.1 Instrument type: Confocal fluorescence microscope, two-photon fluorescence microscope, fluorescence lifetime imaging microscope.
5.2 Ex/Em wavelengths:
5.2.1 One-photon excitation: λₑₓ = 405 nm; dual emission: λₑₘ = 470 nm (green fluorescence for lipid droplets), λₑₘ = 670 nm (red fluorescence for nucleus).

5.3 Result analysis:
5.3.1 Fluorescence intensity changes: In non-ferroptotic cells, green fluorescence intensity (lipid droplets) is high and cytoplasmic red fluorescence intensity is low; during ferroptosis, green fluorescence intensity from lipid droplets decreases, cytoplasmic red fluorescence intensity slightly increases, and the ratiometric value (I₄₇₀ₙₘ/I₆₇₀ₙₘ) decreases.
5.3.2 Fluorescence localization: Green fluorescence localizes to lipid droplets; red fluorescence localizes to the nucleus.
5.3.3 Fluorescence lifetime changes: In non-ferroptotic cells, lipid droplets show a fluorescence lifetime of ~1.46 ns, and nucleus shows ~2.01-2.11 ns; during ferroptosis, lipid droplet fluorescence lifetime decreases to ~0.77 ns while nucleus lifetime remains unchanged; during apoptosis, nucleus lifetime increases while lipid droplet lifetime shows no obvious change.
5.3.4 Polarity-related changes: Green fluorescence from lipid droplets shifts to increased red fluorescence in cytoplasm as lipid droplet polarity increases during ferroptosis; ratiometric images show a polarity gradient from low (red) to high (blue), with gradient homogenization over ferroptosis progression.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.