HMRhoNox-M

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

1. Stock Solution Preparation
1.1 Solvents: DMF; DMSO.
1.2 Recommended concentration: 1 mM.

2. Working Solution Preparation
2.3 Diluents: 50 mM HEPES buffer (pH 7.4); cell culture medium; Hank’s Balanced Salt Solution (HBSS) without phenol red, calcium chloride, and magnesium chloride.
2.2 Working concentration: 1-3 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Adherent cells^[1][2][4]: Including HepG2, SH-SY5Y neuroblastoma cells, and human fibrosarcoma (HT1080) cells.
3.1.2 Bone marrow-derived macrophages (BMDMs, suspension cells)^[3].
3.2 Incubation Conditions
3.2.1 Adherent cells:
3.2.1.1 Incubate with 1-3 μM HMRhoNox-M at 37°C for 30 min.
3.2.1.2 Detection of endogenous Fe²⁺ baseline^[1]: Incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
3.2.1.3 Detection of exogenous Fe^2+[1]: First add 100 μM Fe²⁺ to the cells, incubate at 37°C for 30 min, then incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
3.2.1.4 Monitoring of transferrin-induced iron uptake^[1]: First add 5 μM holoTf to the cells, incubate at 37°C for 30 min, then incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
3.2.1.5 Ferroptosis imaging studies^[4]: Incubate with 1 μM HMRhoNox-M at 37°C for 1 h.
3.2.2 BMDMs^[3]: First pre-incubate the cells with 100 μM ammonium iron citrate for 2 h, then stain with HMRhoNox-M to detect lysosomal Fe²⁺.
3.3 Washing Steps
3.3.1 Adherent cells^[4]: Wash the cells twice with HBSS; for Fe (II) uptake assays, first wash with FBS-free MEM, then wash twice with HBSS.

4. Control Setup
4.1 Negative Controls
4.1.1 Incubate cells with 1 μM HMRhoNox-M and 1 mM 2,2'-bipyridine (Bpy) simultaneously at 37°C for 30 min.
4.1.2 First add 5 μM holoTf + 25 μM apoTf to the cells, incubate at 37°C for 30 min, then incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
4.1.3 First add 5 μM holoTf to the cells, incubate at 4°C for 30 min, then incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
4.1.4 First add 5 μM holoTf + 1 mM NaN₃ to the cells, incubate at 37°C for 30 min, then incubate with 1 μM HMRhoNox-M at 37°C for 30 min.
4.1.5 MCOLN1-knockout BMDMs serve as the negative control for TRPML1-mediated lysosomal Fe²⁺ release.
4.2 Positive Controls
4.2.1 WT BMDMs serve as the positive control for lysosomal Fe²⁺ release^[3].
4.3 Blank Control, used to exclude reagent fluorescence interference^[4].
4.3.1 For ferroptosis imaging studies, set up vehicle-only control group, Erastin (HY-15763)-treated control group, and Erastin + Deferoxamine mesylate (HY-B0988) co-treated control group^[4].

5. Detection and Analysis
5.1 Instrument Types: Fluorescence microscope; fluorescence imaging system.
5.2 Excitation/Emission Wavelengths: Excitation wavelengths are 532-554 nm, 550 nm, or 555 nm; emission wavelengths are 572-642 nm, 575 nm.
5.3 Result Analysis
5.3.1 Changes in Fluorescence Intensity:
5.3.1.1 When intracellular Fe²⁺ levels increase, significant fluorescence turn-on enhancement is observed; in cells where Fe²⁺ is chelated or endocytosis is inhibited, fluorescence intensity decreases to the basal level^[1].
5.3.1.2 Compared with wild-type cells, lysosomal fluorescence intensity is elevated in VPS35-knockdown cells; treatment with R55 reduces the fluorescence intensity of VPS35-knockdown cells^[2].
5.3.1.3 HMRhoNox-M fluorescence intensity decreases in LPS-stimulated WT BMDMs, indicating lysosomal Fe²⁺ release; co-treatment with NAC inhibits this decrease in fluorescence intensity^[3].
5.3.1.4 Fluorescence intensity increases when labile Fe (II) levels rise^[4].
5.3.1.5 In Erastin-induced ferroptosis, lysosomal fluorescence intensity increases at 3 h and 5 h after treatment, and this phenomenon is inhibited by co-treatment with Deferoxamine mesylate^[4].
5.3.2 Fluorescence Localization: Shows a punctate staining pattern, localized to lysosomes; lysosomal membrane^[1][2][3][4].
5.3.3 Color Change: Yellow fluorescence emission; orange-yellow fluorescence^[1][2][4].