Lucifer Yellow CH dipotassium is a high-intensity fluorescent probe containing a free hydrazine group. Lucifer Yellow CH reacts with aliphatic aldehydes at room temperature. Lucifer Yellow CH can be used as a biotracer for monitoring neuronal branching, regeneration, gap junction detection and characterization, and selective ablation of cells after aldehyde fixation. The excitation/emission maxima of Lucifer yellow CH are 430/540 nm respectively.

Physicochemical Properties

Molecular Formula	C13H9N5O9S2-2.2[K+]
Molecular Weight	521.56526
Exact Mass	520.912
CAS #	71206-95-6
Related CAS #	Lucifer Yellow CH dilithium salt;67769-47-5
PubChem CID	12087397
Appearance	Yellow to orange solid
LogP	1.376
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	0
Heavy Atom Count	31
Complexity	914
Defined Atom Stereocenter Count	0
SMILES	O=C1N(NC(NN)=O)C(=O)C=2C3=C(C(=C(C=C13)S(=O)(O)=O)N)C=C(C2)S(=O)(O)=O.[K].[K].[H].[H]
InChi Key	HCWYSCVNSCIZCN-UHFFFAOYSA-L
InChi Code	InChI=1S/C13H11N5O9S2.2K/c14-10-5-1-4(28(22,23)24)2-6-9(5)7(3-8(10)29(25,26)27)12(20)18(11(6)19)17-13(21)16-15;;/h1-3H,14-15H2,(H2,16,17,21)(H,22,23,24)(H,25,26,27);;/q;2*+1/p-2
Chemical Name	dipotassium;6-amino-2-(hydrazinecarbonylamino)-1,3-dioxobenzo[de]isoquinoline-5,8-disulfonate
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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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

Targets	Fluorescent dye/probe
ln Vitro	Making the working solution of Lucifer Yellow CH (dilithium salt). 1. Preparation of stock solution To make a 1 mg/mL stock solution of Lucifer Yellow CH (dilithium salt), dissolve 1 mg of Lucifer Yellow CH (dilithium salt) in 1 mL of DDH2O. Note: After aliquots, the Lucifer Yellow CH (dilithium salt) storage solution should be kept in the dark at -20°C or -80°C. 2. Utilizing the pre-heated serum-free media or PBS, dilute the stock solution into 0.5–1% Lucifer Yellow CH (dilithium salt) working solution. Note: Before using, please make sure that the concentration of the Lucifer Yellow CH (dilithium salt) working solution is appropriate for the current circumstances. Staining of cells 1. Preparation of cells Suspension cells: Centrifuge cells to collect them, then add PBS and wash twice for five minutes each time. Adherent cells: Add islet digested cells after discarding the culture medium. Centrifuge, remove supernatant, add PBS, and wash twice for five minutes each time. 2. Add 1 mL of the working solution for 5–30 minutes (Lucifer Yellow CH, dilithium salt). 3. Discard the supernatant after centrifuging at 400 g for three to four minutes at 4°C. 4. To wash the cells twice, five times apiece, add PBS. 5. Re-suspend the cells in 1 milliliter of PBS or serum-free water, and observe using a flow cytometer or fluorescence microscope. Note: 1. After aliquoting, the Lucifer Yellow CH (dilithium salt) storage solution should be kept out of the light at -20°C or -80°C. Frequent freezing and thawing should be avoided. It can be kept for one month at -20°C and six months at -80°C. 2. You may adjust the Lucifer Yellow CH (dilithium salt) working solution concentration according to your specific needs. 3. This product should not be used for clinical diagnosis or treatment, nor should it be included into food or medication. It is intended only for research use. 4. Please wear disposable gloves and a lab coat for your health and safety.
Enzyme Assay	Contrasting observations exist which indicate that in plants the fluorescent dye lucifer yellow CH (LYCH) either can be used as a tracer for endocytosis or as a substrate for an anion transporter located at the vacuolar membrane. In addition, LYCH as a disulphonated substance may represent an analogue of sulphonated or sulfated natural compounds like some flavonoids. We performed uptake experiments with LYCH into isolated rye vacuoles and observed saturable (Km = 0.3-0.6 mM) vacuolar transport and accumulation of the dye against the concentration gradient only when MgATP was present. GTP and, to a low extent, UTP could substitute for ATP, while the non-hydrolysable ATP analogue AMP-PNP did not drive LYCH uptake. Vanadate and probenecid, the latter substance is known to inhibit organic anion transport at the liver canalicular membrane, both strongly decreased the vacuolar uptake of LYCH, while bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, had no effect. Together with the fact that abolishment of the delta pH via CCCP had only a weak influence on LYCH accumulation, our results indicate that this compound is taken up into rye vacuoles by a directly energized process. Uptake of LYCH was strongly inhibited by other sulfated compounds including sulfobromophthalein and the flavones apigenin 7,4'-disulfate and luteolin 7,4'-disulfate arguing for the presence of a vacuolar transporter for structurally different sulphonated or sulfated compounds. Glucuronates like the rye-specific flavone luteolin 7-O-diglucuronide also strongly decreased uptake of the dye, whereas only a weak effect was observed in the presence of glutathione and a glutathione conjugate, suggesting that LYCH uptake is not mediated via the vacuolar glutathione conjugate pump.
References	[1]. Transport of lucifer yellow CH into plant vacuoles--evidence for direct energization of a sulphonatedsubstance and implications for the design of new molecular probes. FEBS Lett. 1997 Dec 22;420(1):86-92.

Solubility Data

Solubility (In Vitro)

DMSO : ~83.33 mg/mL (~159.77 mM) H2O : ~5 mg/mL (~9.59 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	1.9173 mL	9.5864 mL	19.1729 mL
	5 mM	0.3835 mL	1.9173 mL	3.8346 mL
	10 mM	0.1917 mL	0.9586 mL	1.9173 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.