LHA675

Operating Instructions
(The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)

1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are commonly used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is recommended to prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Phosphate buffer (50 mM, pH = 7.4) containing 10% MeCN.
2.2 Working Concentration: 5 μM.
2.3 Notes: The concentration of the working solution can be adjusted as needed; it must be prepared and used immediately.

3. Staining Procedure
3.1 Sample Type: Adherent cells (HeLa cells, HT22 cells)^[1]
3.1.1 Adherent cells: No trypsin digestion is required for wash-free imaging.
3.2 Incubation Conditions: Incubate with 5 μM LHA675 for 10 min; excitation wavelength λex = 647 nm, emission wavelength λem = 663-738 nm.
3.3 Washing Step: No washing step is required for wash-free imaging.

4. Control Setup
4.1 Positive Control: Cells treated with ferroptosis inducers, including Erastin (HY-15763) (10 μM, 8 h), RSL3 (HY-100218A) (2 μM, 8 h), FIN56 (HY-103087) (5 μM, 10 h).
4.2 Negative Control: Cells pretreated with the ferroptosis inhibitor Ferrostatin-1 (HY-100579) (2 μM, 8 h pretreatment), followed by treatment with Erastin (10 μM, 8 h).
4.3 Blank Control: Untreated cells.

5. Detection and Analysis
5.1 Instrument: Confocal microscope.
5.1.1 Excitation/Emission Wavelengths: λex = 647 nm, λem = 663-738 nm.
5.2 Result Analysis:
5.2.1 Changes in Fluorescence Intensity: After reaction with oxidative free radicals (ROO·, RO·, HO·, ONOO−), fluorescence is significantly turned on (up to 360-fold); fluorescence intensity increases in a dose-dependent manner with the concentration of ferroptosis inducers; the fluorescent signal appears earlier during ferroptosis compared to the gold standard probe.
5.2.2 Fluorescence Localization: The generated fluorophore localizes to mitochondria.
5.2.3 Color Change: The probe emits deep red fluorescence upon activation.

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