Aldehydes, ketones, and carbohydrates via hydrazone formation; metal ions (Cu2+, Hg2+) via chelation; and pH changes via ring-opening of the spirolactam form. Rhodamine 6G hydrazide does not have a single biological target but can be used to label molecules containing carbonyl groups. The hydrazide group reacts with aldehydes and ketones under mildly acidic conditions to form hydrazones, providing a covalent linkage for fluorescent labeling. In the presence of certain metal ions (e.g., Cu2+, Hg2+), the dye can undergo ring-opening of the spirocyclic form, leading to a large increase in fluorescence. This makes it a sensitive “turn-on” fluorescent probe for metal ion detection. Similarly, changes in pH can cause the ring-opening of the spirolactam, and the dye can be used as a pH indicator in the acidic to neutral range (fluorescence increases as pH decreases). In cells, the dye may be used to detect changes in pH or the presence of metal ions. It can also be conjugated to aldehyde-containing biomolecules (e.g., glycoproteins, oxidized carbohydrates) for cellular imaging or biochemical assays. The dye does not have a specific affinity for a particular protein, but it can be used to label proteins that have been modified to contain an aldehyde group (e.g., via periodate oxidation of glycans or via genetic incorporation of aldehyde tags). It is also used in sensing applications for reactive carbonyl species (e.g., methylglyoxal) in biological samples.

Advice (This is our suggested protocol, which should be adjusted to suit your particular requirements as it simply offers guidance) [1]. 1. Prepare 1.0 × 10-3 M of rhodamine 6G hydrazide in methanol. 2. To get the rhodamine 6G hydrazide and sample at workable amounts, dilute them. 3. Fill a quartz cuvette with 2 mL of rhodamine 6G hydrazide (2 × 10 4 M) and top it over with an equal volume of sample solution. 4. Note the spectral information (Em=505-100 nm, Ex=500 nm).

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In vitro, Rhodamine 6G hydrazide is used as a fluorescent probe for the detection of metal ions. At concentrations of 1-10 uM, the dye exhibits very weak fluorescence in the absence of metal ions due to the spirolactam ring being closed. Upon addition of Cu2+ or Hg2+ ions (0.1-100 uM), the dye undergoes ring-opening, resulting in a dramatic increase in fluorescence (excitation 500-530 nm, emission 540-580 nm). The fluorescence intensity is proportional to the metal ion concentration, allowing quantitative detection with high sensitivity (detection limits in the nanomolar to low micromolar range). The probe shows selectivity for Cu2+ and Hg2+ over other metal ions (e.g., Na+, K+, Ca2+, Mg2+, Zn2+, Fe2+, Fe3+), making it useful for selective sensing in biological and environmental samples. The probe can also be used to detect pH changes: at low pH (acidic conditions, pH 2-5), the dye is in the open, fluorescent form; at neutral to basic pH (pH 6-8), it exists predominantly in the non-fluorescent closed spirolactam form. This pH dependence can be exploited to measure intracellular pH changes in live cells. Additionally, the hydrazide group can be used to label aldehyde-containing compounds (e.g., reducing sugars, aldehydes) in solution or on surfaces. For example, the probe can be reacted with an aldehyde-containing small molecule or protein in the presence of a reducing agent (e.g., NaCNBH3) to form a stable fluorescent conjugate. This property is useful for labeling glycoproteins after periodate oxidation to generate aldehydes. In cell-based assays, the dye can be used to detect changes in cellular pH, metal ion levels, or the presence of reactive carbonyl species. The dye is cell-permeable and can be loaded into cells by simple incubation (1-10 uM for 15-60 minutes). After uptake, the dye may localize in lysosomes or other acidic compartments, where it emits fluorescence, providing a means to monitor lysosomal pH or function. It can also be used to track endocytosis and vesicular trafficking. Due to its bright fluorescence and good photostability, it is suitable for live-cell imaging and flow cytometry. The dye may also be used to label membrane proteins or to study protein-protein interactions through aldehyde-tagging strategies. Detailed in vitro protocols are available in the product literature and primary research papers.

In vivo applications of Rhodamine 6G hydrazide are limited, but the dye can be used for imaging in small animal models, particularly for sensing pH or metal ions. For example, the probe can be injected intravenously (tail vein) into mice at a dose of 0.1-1 mg/kg in PBS/<10% DMSO. The dye may accumulate in tumors or sites of inflammation due to the acidic microenvironment (pH ~6.5-6.8) and produce a “turn-on” fluorescence signal. Alternatively, it can be used to label aldehyde-bearing molecules (e.g., nanoparticles, antibodies) for targeted imaging. However, the green to yellow fluorescence emission (550-580 nm) has limited tissue penetration, and autofluorescence from tissues may interfere with deep imaging. The probe is also used for ex vivo imaging of tissue sections or whole organs after systemic administration. It has potential applications for detecting copper or mercury poisoning in animal models, but these are experimental. The dye is not approved for clinical use. Researchers should be cautious when administering the dye in vivo due to its potential toxicity at high doses and the lack of extensive safety data. For most applications, the dye is used for in vitro or ex vivo analysis.

For a non-cellular metal ion sensing assay, Rhodamine 6G hydrazide is dissolved in DMSO to prepare a 1-10 mM stock solution. The stock solution is diluted to a final concentration of 1-10 uM in a suitable buffer (e.g., 10 mM HEPES pH 7.4, or 10 mM Tris-HCl pH 7.4, or PBS, pH 7.4) containing up to 1% DMSO. A series of metal ion solutions (e.g., CuCl2, HgCl2, ZnCl2, FeSO4, NaCl, KCl, CaCl2, MgCl2) are prepared in the same buffer at concentrations ranging from 0.1 uM to 100 uM. In a 96-well black plate, 100 uL of the probe solution is added to each well, followed by 100 uL of the metal ion solution (or buffer alone for blank). The plate is incubated for 5-30 minutes at room temperature. Fluorescence intensity is measured using a fluorescence plate reader with excitation at 500-530 nm and emission at 540-580 nm (e.g., Ex=525 nm, Em=555 nm). A significant increase in fluorescence is observed in the presence of Cu2+ or Hg2+, while other metal ions cause little or no change. The selectivity of the probe for the target metal ion can be assessed by comparing the fluorescence enhancement ratio (F/F0) for each ion. For calibration, a standard curve of fluorescence intensity vs. metal ion concentration is plotted, and the limit of detection (LOD) is calculated as 3σ/slope, where σ is the standard deviation of the blank. The assay can be performed in a cuvette using a fluorimeter as well. For pH sensing, the dye (1-10 uM) is prepared in buffers of varying pH (e.g., acetate buffer pH 3-5, phosphate buffer pH 6-8, Tris-HCl pH 7-9), and fluorescence is measured (Ex/Em 500-530/540-580 nm). Fluorescence is highest at low pH (open form) and lowest at neutral/alkaline pH (closed form). The pKa can be estimated by fitting the fluorescence change vs. pH to a sigmoidal curve. For labeling aldehyde-containing molecules, the probe is dissolved in methanol or DMSO, then diluted to 10-100 uM in acetate buffer (pH 4.5-5.5) or in a suitable organic solvent. The aldehyde-containing compound (e.g., an aldehyde-tagged protein, a reducing sugar, or a small molecule aldehyde) is added to the probe solution. The reaction is allowed to proceed for 1-4 hours at room temperature or 37degC. If desired, a reducing agent (e.g., 1-10 mM NaCNBH3) can be added to reduce the hydrazone to a stable amine linkage. The labeled product can be purified by chromatography (e.g., SEC, HPLC) or dialysis. Labeling efficiency is assessed by measuring the absorbance of the dye (e.g., at 530 nm for rhodamine 6G) and the protein concentration (A280) or by using a fluorescence plate reader.

A typical in vitro cellular imaging protocol for Rhodamine 6G hydrazide is as follows. Cells (e.g., HeLa, CHO, macrophages) are seeded on coverslips or in 96-well plates (1×10⁴ cells/well) and cultured overnight. A stock solution of R6GH is prepared in DMSO (1-10 mM). For staining, the stock is diluted to a final concentration of 1-10 uM in serum-free cell culture medium (or HBSS) with a final DMSO concentration ≤0.5%. The culture medium is removed from the cells, and the cells are washed once with PBS. The dye solution is added to the cells, and the cells are incubated for 15-60 minutes at 37degC in a humidified incubator (5% CO2). After incubation, the dye solution is removed, and the cells are washed 2-3 times with PBS to remove extracellular dye. For live-cell imaging, the cells are placed in fresh, phenol red-free medium and imaged immediately using a fluorescence microscope with an appropriate filter set (e.g., Ex 530-550 nm, Em >570 nm for rhodamine 6G, or FITC filter Ex 488 nm, Em 505-550 nm? Actually, rhodamine 6G has Ex/Em around 530/550 nm, so TRITC or Cy3 filter sets are appropriate: Ex 540-560 nm, Em 570-600 nm). For fixed-cell imaging, cells can be fixed with 4% paraformaldehyde for 10-15 minutes at room temperature after staining, washed, and mounted with anti-fade mounting medium. For detection of metal ions in cells, cells can be pre-incubated with the metal ion (e.g., 10-100 uM Cu2+ or Hg2+) for 1-24 hours, then washed and stained with R6GH (1-10 uM) for 30-60 minutes, then washed and imaged. An increase in intracellular fluorescence indicates the presence of the metal ion. For pH sensing, cells can be incubated in buffers of different pH in the presence of the dye, and the fluorescence intensity is quantified. For flow cytometry, cells are stained in suspension (1×10⁶ cells/mL) with 1-10 uM R6GH for 30 minutes, washed, and analyzed using a flow cytometer with a 488 nm laser (or 532 nm laser) and an emission filter of 570-610 nm (PE channel). The dye may also be used to label surface glycoproteins after periodate oxidation to generate aldehydes: cells are treated with 1-10 mM sodium periodate in PBS for 10-30 minutes on ice, washed, then reacted with 10-100 uM R6GH in acetate buffer pH 5.5 for 1-2 hours at room temperature, followed by washing and imaging. The hydrazone bond can be reduced with NaCNBH3 for stability if needed. The compound is for research use only, and protective measures should be taken to avoid skin/eye contact and inhalation.

An in vivo protocol for sensing metal ions or pH could involve the following steps (for research purposes only). Rhodamine 6G hydrazide is dissolved in sterile PBS containing 5-10% DMSO and 10% Tween-80 to a final concentration of 0.5-2 mg/mL. The solution is filtered through a 0.22 um filter. Mice (e.g., 6-8 week old, 20-25 g, n=5 per group) are injected intravenously (tail vein) with 100-200 uL of the solution (dose 1-10 mg/kg). For metal ion imaging, a model of copper overload or mercury poisoning can be induced by intraperitoneal injection of CuCl2 (e.g., 5-10 mg/kg) or HgCl2 (1-5 mg/kg) 1-24 hours before the dye injection. After dye injection, mice are imaged at various time points (e.g., 30, 60, 120 minutes) using a whole-body fluorescence imaging system with excitation at 530-550 nm and emission at 570-600 nm. Alternatively, mice are euthanized, and organs (liver, kidney, spleen, lung, heart, brain) are harvested for ex vivo fluorescence imaging and tissue section analysis. For pH imaging, a tumor-bearing mouse model (e.g., xenograft with acidic microenvironment) can be used. The dye is injected intravenously, and fluorescence images are acquired to detect the acidic tumor core. However, due to the green-yellow emission, penetration depth is limited (a few mm), so this approach is best for superficial tumors or ex vivo analysis. Researchers are advised to validate the in vivo performance with their own pilot studies, as detailed published in vivo protocols for R6GH are not extensive. The dye is not approved for human use; these procedures are for research purposes only.

The pharmacokinetics of Rhodamine 6G hydrazide (MW 428.52, logP ~2-3) have not been extensively reported. Based on its structure and the properties of similar rhodamine dyes, the compound is likely to have a rapid distribution phase after intravenous administration, with a half-life of minutes. It is likely taken up by the liver and spleen and may accumulate in the kidneys. The unmodified hydrazide may be metabolized in the liver, and the metabolites may be excreted in the urine and bile. The “turn-on” property of the dye in the presence of metal ions or low pH complicates the interpretation of PK data, as the fluorescence signal is not directly proportional to the concentration of the intact dye. For quantitative analysis, the dye can be extracted from plasma or tissues by organic solvent (e.g., acetonitrile or methanol) and analyzed by HPLC with fluorescence detection or by LC-MS/MS. However, detailed PK parameters (Cmax, Tmax, AUC, t1/2, Vd, CL) are not commonly reported in the literature, as the compound is primarily used as a sensing probe rather than a therapeutic agent. Researchers needing PK data are encouraged to perform custom studies or consult the primary literature for specific applications.

Toxicological data for Rhodamine 6G hydrazide are limited. It is considered a research chemical and should be handled with caution. The dye may be moderately toxic if ingested, inhaled, or absorbed through the skin. It may cause eye, skin, and respiratory tract irritation. In vitro, the dye has been used at concentrations of 1-10 uM without acute toxicity in several cell lines (e.g., HeLa, HEK293) over short-term incubation (1-2 hours). At higher concentrations (e.g., >50 uM) or longer incubation (>24 hours), it may cause cell death, possibly due to disruption of mitochondrial function or oxidative stress. In vivo, the acute toxicity has not been fully characterized. Other rhodamine dyes (e.g., rhodamine 6G, rhodamine B) have known toxicities at high doses, including hepatotoxicity and nephrotoxicity. R6GH should be handled with appropriate personal protective equipment (gloves, lab coat, eye protection). Work in a well-ventilated area or fume hood when handling powders. The compound should be stored at -20degC, protected from light, and away from sources of ignition. It is for research use only and is not intended for human clinical use. Dispose of waste according to local regulations.

[1]. Optical sensing of hydrogen sulphate using rhodamine 6G hydrazide from aqueous medium. Spectrochim Acta A Mol Biomol Spectrosc. 2017 Jun 5;180:44-50.

Rhodamine 6G hydrazide (R6GH) is a research-grade fluorescent dye with no approved clinical applications. It is supplied as a solid powder (color: likely red to purple) with a purity of ≥98% (typically >98%). The molecular weight is 428.52, and the formula is C26H28N4O2. The dye is soluble in DMSO and methanol, and moderately soluble in water (may require sonication). It should be stored at -20degC, protected from light, and in a dry environment. Stock solutions (1-10 mM) in DMSO can be stored at -20degC for up to 6 months, but repeated freeze-thaw cycles should be avoided. R6GH is widely used in selective colorimetric and fluorescent sensing for metal ions (Cu2+, Hg2+), pH, and reactive carbonyl species. It is also used for labeling aldehyde-containing compounds and for cellular imaging of metal ions, pH changes, and glycoproteins. The hydrazide group confers reactivity with aldehydes and ketones, allowing bioconjugation applications. The dye is cell-permeable and can be used in live-cell imaging. It is also used in studies of cellular processes, drug delivery, and protein-protein interactions due to its vivid fluorescence and reactivity. Synonyms include R6GH and possibly rhodamine 6G hydrazone. For detailed protocols, users are encouraged to consult the product data sheet and primary literature. This compound is for research use only and is not for diagnostic or therapeutic use. Ensure personal safety while handling.

C26H28N4O2

428.53

428.221

932013-08-6

71004110

Off-white to pink solid powder

1.30±0.1 g/cm3 (20 °C, 760 mmHg)

623.4±65.0 °C (760 mmHg)

5.678

3

5

4

32

667

0

CCNC1=CC2=C(C=C1C)C3(C4=CC=CC=C4C(=O)N3N)C5=C(O2)C=C(C(=C5)C)NCC

QUMMHDKUYXGXQJ-UHFFFAOYSA-N

InChI=1S/C26H28N4O2/c1-5-28-21-13-23-19(11-15(21)3)26(18-10-8-7-9-17(18)25(31)30(26)27)20-12-16(4)22(29-6-2)14-24(20)32-23/h7-14,28-29H,5-6,27H2,1-4H3

2-amino-3',6'-bis(ethylamino)-2',7'-dimethylspiro[isoindole-3,9'-xanthene]-1-one

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~10 mg/mL (~23.34 mM)

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)