DRhFe

Guidelines (The following is a recommended experimental protocol for reference only; please adjust according to actual needs)

1. Stock Solution Preparation
1.1 Solvent: anhydrous DMSO
1.2 Recommended concentration: 1 mM

2. Working Solution Preparation
2.1 Diluent: DMEM or PBS supplemented with 10% FBS.
2.2 Working concentration: 10 μM.
2.3 Notes: Adjust the working concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Adherent cells^[1]:
3.1.1 No trypsin digestion is required for staining or imaging.
3.1.2 Incubation conditions: incubate with 10 μM DRhFe at 37°C for 30 min.
3.1.3 Washing steps: rinse 2 times with PBS after staining; after pretreatment with ferroptosis inducers/inhibitors, rinse 3 times with PBS. (Note: Some alternative protocols may omit the washing steps.)
3.2 Fresh rat kidney sections^[1]:
3.2.1 Prepare using a vibratome.
3.2.2 Incubation conditions: incubate with 10 μM DRhFe at 8°C for 2 h.
3.2.3 Washing steps: after pretreatment with Ferric citrate (HY-N1428C), rinse 3 times with PBS.

4. Control Setup
4.1 Negative control: adherent cells treated with medium only; tissue sections incubated with DRhFe working solution only; adherent cells stained with 10 μM DRhFe only.
4.2 Ferroptosis inhibition control: adherent cells co-incubated with ferroptosis inducer + iron chelator, ferroptosis inducer + ferroptosis inhibitor; adherent cells pretreated with 1 μM Erastin (HY-15763) and 100 μM DFO (HY-D0903) for 8 h, followed by staining with 10 μM DRhFe; adherent cells pretreated with 1 μM erastin and 1 μM Ferrostatin-1 (HY-100579) for 8 h, followed by staining with 10 μM DRhFe.
4.3 Fe³⁺ regulation control: adherent cells pre-incubated with 20 μM Fe³⁺ for 8 h, followed by staining with 10 μM DRhFe; adherent cells pre-incubated with 20 μM Fe³⁺ for 8 h, then treated with 20 μM TPEN for 0.5 h, followed by staining with 10 μM DRhFe.
4.4 Ferroptosis induction control: adherent cells pretreated with 1 μM Erastin for 8 h, followed by staining with 10 μM DRhFe.
4.5 Positive control: used to validate the effectiveness of the experimental system.
4.6 Blank control: to exclude autofluorescence interference from reagents.

5. Detection and Analysis
5.1 Instrument type: laser confocal scanning microscope; confocal fluorescence microscope.
5.2 Excitation/emission wavelengths:
5.2.1 Green channel:
- Ex = 405 nm, Em = 440−500 nm (for adherent cells).
- Ex = 405 nm, Em = 440−530 nm (for tissue samples).
5.2.2 Red channel:
- Ex = 561 nm, Em = 570−630 nm (for adherent cells).
- Ex = 552 nm, Em = 560−650 nm (for tissue samples).
5.3 Result analysis:
5.3.1 Fluorescence intensity changes: In the presence of Fe³⁺, red channel fluorescence increases; green channel fluorescence may decrease or show no significant decrease, resulting in an elevated F_red/F_green ratio.
5.3.2 Fluorescence localization: In HeLa cells, DRhFe mainly localizes to the endoplasmic reticulum and lysosomes.
5.3.3 Color change: Fluorescence shifts from green (free DRhFe) to red (DRhFe-Fe³⁺ complex); cells stained with DRhFe alone exhibit green fluorescence, and red fluorescence intensity increases with increasing Fe³⁺ concentration.
5.3.4 Ratiometric analysis: Calculate the mean F_red/F_green ratio to quantify labile Fe³⁺ levels.

DRhFe (up to 40 μM; 24 h) exhibits low cytotoxicity in HeLa cells, demonstrating good biocompatibility^[1].