IOPBA

Operating Instructions (The following is a recommended experimental protocol for guidance only; please adjust according to actual requirements)
1. Stock Solution Preparation
1.1 Solvent: 0.2 M HEPES buffer (pH 7.4) containing 2 vol% DMSO.
1.2 Concentration recommendation: 0.1 mM.
2. Working Solution Preparation
2.1 Diluent: 0.2 M HEPES buffer (pH 7.4) containing 2 vol% DMSO.
2.2 Working concentrations: 10 μM, 15 μM, and 20 μM for cell imaging; the iron ion concentration for cell imaging is 1 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare it immediately before use.
3. Staining Procedure
3.1 Sample type: Adherent cells (HeLa cells)
3.1.1 Adherent cells: No trypsin digestion is required before staining.
3.2 Incubation conditions:
3.2.1 Incubate HeLa cells with 10 μM, 15 μM, or 20 μM IOPBA at 37°C under 5% CO₂ for 30 min.
3.2.2 Iron ion detection: Incubate IOPBA-stained cells with 1 μM iron ions at 37°C under 5% CO₂.
3.2.3 Fluoride ion detection: Incubate iron ion-IOPBA-stained cells with fluoride ions at 37°C under 5% CO₂.
3.3 Washing step: After IOPBA incubation, wash the cells once with Dulbecco's Phosphate-Buffered Saline (DBS).
4. Detection and Analysis
4.1 Instrument: Laser confocal scanning microscope.
4.1.1 Excitation wavelength: 342 nm; emission wavelength: 458 nm.
4.2 Result analysis:
4.2.1 Fluorescence intensity change: Cells stained with IOPBA exhibit strong blue fluorescence; the fluorescence intensity decreases with increasing iron ion concentration, and recovers after the addition of fluoride ions.
4.2.2 Fluorescence localization: The blue fluorescence distributes throughout the cytoplasm of HeLa cells.
4.2.3 Color change: Strong blue fluorescence (IOPBA only) → Weak blue/quenched fluorescence (IOPBA + iron ions) → Recovered strong blue fluorescence (IOPBA + iron ions + fluoride ions).

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