FlCFe1

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


1. Staining Procedure^[1]
1.1 Sample Types:
1.1.1 Adherent cells: DLD-1 colorectal adenocarcinoma cells.
1.1.2 3D multicellular model: DLD-1 tumor spheres (10⁶ cells).
1.2 Incubation Conditions:
1.2.1 Adherent cells: Co-incubate with 10 μM FlCFe1 for 2 h.
1.2.2 Tumor spheres: Co-incubate with 10 μM FlCFe1 for 24 h.

2. Control Setup
2.1 Set up vehicle control groups for both adherent cells and tumor spheres.
2.2 Set up iron depletion control group: After incubation with FlCFe1, pretreat adherent cells with salicylaldehyde isonicotinoyl hydrazone (SIH) for 30 min.
2.3 Set up iron supplementation control group: After incubation with FlCFe1, pretreat adherent cells with ammonium ferrous sulfate (FAS) for 30 min.

3. Detection and Analysis
3.1 Instrument: Confocal fluorescence microscope
3.1.1 Excitation wavelength: 405 nm; emission wavelengths: 425-480 nm (blue channel) and 520-600 nm (yellow channel).
3.2 Result Analysis:
3.2.1 Changes in fluorescence intensity: A higher blue/yellow emission ratio indicates a higher level of labile iron pool (LIP), while a lower ratio indicates a lower LIP level.
3.2.2 Fluorescence localization: Localizes to the cytoplasm in adherent cells, with no specific organelle targeting.
3.2.3 Penetration in 3D model: FlCFe1 can penetrate tumor spheres up to 200 μm, enabling ratio-based LIP detection throughout the entire tumor sphere.
3.2.4 Response to iron chelator: The iron chelator only reduces the blue/yellow ratio in the approximately 20 μm outer region of tumor spheres.

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