RhoNox-1 is a fluorescent probe that specifically detects divalent iron ions. When RhoNox-1 reacts with Fe2+, it can generate an irreversible orange (red) fluorescent product (Ex/Em: 540/575 nm). FeRhoNox-1 can enter cells very well, is suitable for the detection of Fe2+ in living cells, and tends to be located in the Golgi apparatus.

Physicochemical Properties

Molecular Formula	C28H31N2O4+
Molecular Weight	459.557
Exact Mass	458.22
CAS #	1447815-38-4
PubChem CID	168008618
Appearance	Pink to red solid powder
LogP	4.2
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	900
Defined Atom Stereocenter Count	0
SMILES	C(C1C=CC=CC=1C1=C2C=CC(N(CC)CC)=CC2=[O+]C2C=C(N(=O)(CC)CC)C=CC1=2)(=O)O
InChi Key	VBBOPWUGWHUQLA-UHFFFAOYSA-N
InChi Code	InChI=1S/C28H30N2O4/c1-5-29(6-2)19-13-15-23-25(17-19)34-26-18-20(30(33,7-3)8-4)14-16-24(26)27(23)21-11-9-10-12-22(21)28(31)32/h9-18H,5-8H2,1-4H3
Chemical Name	2-[3-diethylazaniumylidene-6-[diethyl(oxido)azaniumyl]xanthen-9-yl]benzoate
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

1. RhoNox-1 functioning solution components 1.1 To produce the stock solution, use 110 μL of anhydrous DMSO. To obtain a 1 mM stock solution, use 50 μg of RhoNox-1. 1.2 Elements of a functional solution To make a 1-10 μM RhoNox-1 working solution, use serum-free cell bone marrow or PBS storage solution that has already been made. Note: Please prepare RhoNox-1 working fluid for immediate usage by adjusting its concentration to suit the current circumstances. 2. Staining suspension cells with cells 2.1 Suspension cells: Centrifuge the cells to collect them, then add PBS and wash twice for five minutes apiece. At 1×106/mL, the cell density 2.2, after staining, add 1 mL of working solution and wait for 5 to 30 minutes. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or plasma-free solution, and use a flow cytometer or fluorescence microscope to monitor. 3. Staining adherent cells in cells 3.1 Apply stain to the adhering cells. 3.2 Aspirate extra cells and remove the coverslip from the culture medium. 3.3 Cover the cells completely with 100μL of dye working solution and shake gently for a duration of 5-30 minutes. 3.4 Aspirate the dye working solution, give the cells two to three 5-minute washings, and use a flow cytometer or fluorescence microscope to observe.

References

[1]. Histological detection of catalytic ferrous iron with the selective turn-on fluorescent probe RhoNox-1 in a Fenton reaction-based rat renal carcinogenesis model. Free Radic Res. 2014 Sep;48(9):990-5. [2]. Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool. Tumour Biol. 2017 Jul;39(7):1010428317717655. [3]. Contrasting intra- and extracellular distribution of catalytic ferrous iron in ovalbumin-induced peritonitis. Biochem Biophys Res Commun. 2016 Aug 5;476(4):600-606.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~218.08 mM)

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1760 mL	10.8800 mL	21.7599 mL
	5 mM	0.4352 mL	2.1760 mL	4.3520 mL
	10 mM	0.2176 mL	1.0880 mL	2.1760 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.