FerroOrange (Fe2+ indicator)

1. Probe preparation:

1.1 Remove FerroOrange from the refrigerator and allow it to thaw at room temperature. Place it in a low-speed microcentrifuge and centrifuge until the powder in the bottle settles to the bottom, then open the lid.

1.2 Add 35μL of DMSO to the tube containing 24μg of FerroOrange, and mix thoroughly by pipetting up and down until it is completely dissolved to prepare a 1mM FerroOrange storage solution. If the storage solution cannot be used up in one experiment, aliquot and store it in the dark at -20℃ for up to one month.

1.3 Prior to the actual experiment, dilute the 1mM FerroOrange storage solution to the desired working concentration (e.g. 1uM) with neutral buffer or serum-free culture medium. The working solution should be freshly prepared and used as soon as possible.

Note: acidic solutions will oxidize FerroOrange, severely affecting the efficiency of the probe.。

2. Fluorescence microscopy operating procedure:

2.1. Seed cells in a fluorescence culture dish and culture overnight in a 37℃, 5% CO₂ incubator.

2.2. Discard the supernatant and wash cells three times with HBSS or serum-free culture medium.

2.3. Replace with drug-containing culture medium and culture in a 37℃, 5% CO₂ incubator.

Note：Optimize the incubation time based on the characteristics of the drug.

2.4. Add 1 μM working solution of FerroOrange and culture in a 37℃, 5% CO₂ incubator for 30min.

Note：Observe after adding FerroOrange dye, without washing.

2.5. Observe cells under a fluorescence microscope.

3. Operating procedure for flow cytometry:

3.1. Seed 1 x 10⁵ HeLa cells (in MEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) into a 6-well plate and culture overnight in a 37℃, 5% CO₂ incubator.

3.2. Wash cells three times with serum-free culture medium (2 mL).

3.3. Add serum-free culture medium (1 mL) to the control group cells, and add 10 mM ammonium iron sulfate (10μL) (final concentration: 100μM) to the ammonium iron sulfate(II)-treated group cells in serum-free culture medium.

3.4. Incubate cells in a 37℃ incubator for 20 minutes, and wash cells three times with HBSS (1 mL).

3.5. Digest cells with trypsin (250 µL) and stop the reaction with serum-containing culture medium (1 mL), then transfer 1.25 mL of cell suspension to a microcentrifuge tube.

3.6. Centrifuge the cell suspension at 1,500 rpm for 3 minutes.

3.7. Discard the supernatant and add HBSS (1 mL) to the microcentrifuge tube, mix by shaking.

3.8. Centrifuge the cell suspension at 1,500 rpm for 3 minutes, and discard the supernatant.

3.9. Add 1μM FerroOrange and MEM containing serum-free culture medium (300μL) to the cells.

3.10. Incubate the cells at 37℃ for 15-30 minutes.

3.11. Filter cells through a cell strainer, and analyze the sample using a flow cytometer.

4. Fluorescence detection:

4.1. For fluorescence microscopy: use universal G excitation filters such as those used for Cv3 detection.

4.2. For laser microscopy and flow cytometry: use 532nm, 514nm or 561nm lasers for excitation. If not available, a 488nm laser can also be used. The emission wavelength is 580nm.

Fig. 1  FerroOrange staining of HeLa cells treated with ammonium iron sulfate.

5. Operating procedure for fluorescence microplate reader:

5.1. Set up groups:

Sample A: HeLa cells with no additives.

Sample B: HeLa cells with addition of iron chelator 2,2'-bipyridyl (Bpy)（GC61754）.

Sample C: HeLa cells with addition of iron (ammonium iron sulfate)（GC20135）.

5.2. Seed 100 µl of HeLa cell suspension into each well of a 96-well black plate (with a clear bottom) to achieve a density of 10,000 cells/well, and culture overnight in a 37℃, 5% CO₂ incubator.

5.3. Wash the cells in Sample C three times with 100 µl of MEM (without FBS).

5.4. Add 100 µl of ammonium iron sulfate/MEM (without FBS) (final concentration: 100 µM) to Sample C and let it sit for 30 minutes in a 37℃, 5% CO₂ incubator.

5.5. Wash all wells with 100 µl of HBSS three times.

5.6. Add 100 µl of 1 µM FerroOrange working solution to Samples A and C, and add 100 µl of HBSS solution containing FerroOrange (final concentration: 1 µM) and Bpy (final concentration: 100 µM) to Sample B. Incubate all samples for 30 minutes in a 37℃, 5% CO₂ incubator.

5.7. Measure the fluorescence intensity of each sample using a multifunctional microplate reader (Ex: 543 nm, Em: 580 nm).

Fig. 2 Results of fluorescence microplate reader detection.

This protocol only provides a guideline, and should be modified according to your specific needs.