LHA930

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

1. Stock Solution Preparation
1.1 Solvent: In literature, the administration solution of LHA930 for animal experiments is prepared with PBS containing 10% DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Phosphate buffer (50 mM, pH = 7.4) containing 50% MeCN.
2.2 Working Concentration: 5 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh immediately before use.

3. Staining Procedure
3.1 Sample Types: In vitro oxidative free radical reaction system; APAP-induced liver injury model in BALB/c mice.
3.2 Incubation Conditions:
3.2.1 In vitro: LHA930 (5 μM) reacts with oxidative free radicals including peroxyl radical (ROO·), alkoxyl radical (RO·), hydroxyl radical (HO·) and peroxynitrite (ONOO^-) in phosphate buffer (50 mM, pH = 7.4) containing 50% MeCN, and the changes in fluorescence signal are detected.
3.2.2 In vivo: 12 h after APAP administration, LHA930 (1 mg/kg) dissolved in PBS containing 10% DMSO is injected into BALB/c mice via tail vein.

4. Control Setup
4.1 Set up negative control, positive control and blank control.
4.2 Positive Control: BALB/c mice treated with APAP (100, 300 or 500 mg/kg), used to induce ferroptosis-related liver injury of different degrees.
4.3 Negative Control: Mice pretreated with Fer-1 (3 mg/kg/day for 3 consecutive days) before administration of APAP (300 mg/kg).
4.4 Blank Control: Mice injected with PBS.
4.5 Positive control is used to verify the experimental system; blank control is used to eliminate fluorescence interference from reagents.

5. Detection and Analysis
5.1 Instruments: Small animal near-infrared fluorescence imager
5.1.1 In vitro: Fluorescence spectroscopy is used to analyze the fluorescence changes of LHA930 after reaction with oxidative free radicals. LHA930 can respond to oxidative free radicals such as ROO·, RO·, HO· and ONOO^-, and produce a fluorescence enhancement signal.
5.1.2 In vivo: Ex = 880 nm, Em = 1200 nm long-pass filter.
5.2 Result Analysis:
5.2.1 Changes in fluorescence intensity: The liver area of APAP-treated mice shows concentration-dependent and time-dependent fluorescence enhancement; the fluorescence intensity of ferroptosis-induced cells is higher than that of control cells.
5.2.2 Fluorescence localization: Activation mainly occurs in the liver, spleen and intestine in vivo; after activation in vitro, fluorescence is localized in mitochondria.
5.2.3 Fluorescence signal: Near-infrared fluorescence is turned on after reaction with peroxyl radical, alkoxyl radical, hydroxyl radical and peroxynitrite anion.

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