Pyronin Y (PY) is a fluorescent probe that binds to double-stranded RNA (dsRNA). Its targets within cells include various RNA subspecies such as messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). It is also known to localize to mitochondria and Cajal bodies. The binding is based on its interaction with RNA structures rather than a specific protein target.[4]

In order to estimate formalin-fixed EL4 blank tumors, cellular enzyme-dispersed R3327-RNA content per cell in G-transporting adenocarcinoma cells, mouse splenocytes stimulated with concanavalin, and human peripheral blood stimulated with phytohemagglutinin, pyronin Y forms a fluorophore complex with double-stranded sequences, particularly RNA [1]. Give an explanation of the Jionin Y-based fluorescent staining technique. Human reticulocytes have been treated with this method to dye their RNA in preparation for sorting and process analysis [2]. This dye gathers in the mitochondria of live cells. Jiaoning Y forms additional mitochondria in the mitochondria of cultivated cells when it is almost entirely localized there at 1.7 to 3.3 μM! Rhodamine 123 probe. PY is not harmful at this dose, but it will stop cell division and induce apoptosis [3]. For a considerable amount of time, paraffin tissue sections have been differentially stained using pyronin Y in conjunction with other dyes like methyl green. Cell nuclei appeared non-fluorescent in methyl green-pyronin Y stained sections, whereas red blood cells, elastic fibers, amygdala acinar cell zymogen granules, liver cell surface membranes, and renal tubular cells all displayed intense green and/or red fluorescence[4]. Double-stranded RNA (dsRNA) such as messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) are bound by pyrin Y [5].

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In live NIH3T3 cells, Pyronin Y (12 µM) was used to label RNA. Spectral phasor analysis of the fluorescence emission (excitation at 514 nm) revealed at least four distinct PY-labeled species with different spectral properties and spatial distributions: 1) Transcripts within nucleoli, proposed to be ribosomal RNA (rRNA); 2) Clustered cytoplasmic transcripts, proposed to be messenger ribonucleoprotein (mRNP) complexes; 3) Filamentous cytoplasmic structures composed of puncta that localized along mitochondria; 4) Nuclear structures in proximity to nucleoli, proposed to be Cajal bodies.[4]
The average emission wavelength and width for different PY-labeled species were quantified: Nucleolar species had a wavelength of 559.6 ± 2.9 nm and width of 31.6 ± 1.4 nm; Cytoplasmic species had an average wavelength of 555.2 ± 1.7 nm and width of 30.2 ± 1.0 nm; Nuclear species had an average wavelength of 554.8 ± 1.7 nm and width of 36.3 ± 3.1 nm. These spectral differences, though small (<5 nm apart), allowed their discrimination using spectral phasor analysis.[4]
Dual labeling experiments with Pyronin Y (red) and mitochondrial marker mito-eGFP (green) showed that cytoplasmic PY-labeled RNA puncta moved along mitochondrial tracks, indicating an interaction with the mitochondrial matrix, but there was very little colocalization of the two signals.[4]

Live Cell Imaging and Spectral Phasor Analysis: NIH3T3 cells were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Cells were plated onto 35 mm dishes pre-coated with fibronectin (3 µg/mL) 24 hours prior to imaging. At the time of plating, cells were exposed to Pyronin Y at a concentration of 12 µM. Cells were imaged within 5 hours of plating and maintained at 37°C with 5% CO2 during imaging. Fluorescence images were acquired using a confocal microscope in Lambda mode with a 514 nm excitation laser (maximum 9% power). Emission was collected from 416 nm to 728 nm across 32 channels using a 40x water immersion objective. Images were 256 x 256 pixels with a pixel dwell time of 177.85 µs. Z-stacks were acquired with a step size of approximately 1 µm. Spectral phasor analysis was performed on the lambda stack images using custom software (SimFCS). Each pixel's emission spectrum was Fourier-transformed to generate coordinates in a spectral phasor plot. Clusters of pixels with similar spectral properties in the phasor plot were manually selected and mapped back to their spatial locations in the cell image to identify different RNA subspecies and their association with cellular structures (nucleoli, cytoplasm, nucleus, Cajal bodies).[4]
Co-localization with Mitochondria: NIH3T3 cells were co-labeled with mito-eGFP (a mitochondrial-targeted enhanced green fluorescent protein) and Pyronin Y (12 µM). Time-lapse images (512 x 512 pixels) were acquired to visualize the spatial relationship and dynamics between the red PY-labeled RNA puncta and the green mitochondrial network. Images were captured at 6-second intervals.[4]

The article references prior studies (cited as references 1-4 within the provided text) indicating that Pyronin Y can have cytotoxic and cytostatic properties related to its mitochondrial localization and interaction with RNA. However, the current study [4] itself does not provide new quantitative toxicity data (e.g., IC50, LD50) for Pyronin Y under the experimental conditions used (12 µM, 5-hour exposure). It notes that in their experiments, cells were imaged within 5 hours of exposure.[4]

[1]. Flow cytometric analysis of RNA content in different cell populations using pyronin Yand methyl green. Cytometry. 1982 Jul;3(1):28-35.

[2]. Flow cytometry of human reticulocytes based on RNA fluorescence. Cytometry. 1981 Mar;1(5):313-20.

[3]. Cytostatic and cytotoxic properties of pyronin Y: relation to mitochondrial localization of the dye and its interaction with RNA. Cancer Res. 1986 Nov;46(11):5760-6.

[4]. Pyronin Y as a fluorescent stain for paraffin sections. Histochem J. 2002 Jun-Jul;34(6-7):299-303.

[5]. Spectral phasor analysis of Pyronin Y labeled RNA microenvironments in living cells. Biomed Opt Express. 2013 Jan 1;4(1):171-7.

Pyronin Y is an organochlorine salt with a cation of 6-(dimethylamino)-N,N-dimethyl-3H-xanthon-3-imine. When used in combination with methyl green, it selectively displays RNA (red) but not DNA (green) using the Unna-Pappenheim method. It is a histological dye. It is an imine and organochlorine salt containing the pyronin Y cation. Xanthon dyes are used as staining agents for bacteria and biological processes. Synonyms: pyronin; pyronin G; pyronin Y. It can also refer to pyronin B, whose cation is diethyl rather than dimethylamino.

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Pyronin Y is an environment-sensitive fluorescent dye. It is used here as a universal label for double-stranded RNA, allowing for the visualization of overall RNA abundance and distribution in live cells without the need for specific labeling of individual RNA species. The study demonstrates the novel application of spectral phasor analysis to discriminate between different RNA-protein complexes based on small spectral shifts in PY's emission, even when these shifts are less than 5 nm. This method does not require prior knowledge of the individual spectral components. The work suggests that PY can be a complementary tool to specific RNA probes for studying RNA biology in living systems.[4]

C17H19CLN2O

302.7986

302.118

C, 67.43; H, 6.32; Cl, 11.71; N, 9.25; O, 5.28

92-32-0

7085

Light brown to black solid powder

250-260ºC(lit.)

0

3

1

21

480

0

[Cl-].[O+]1C2C(=CC=C(N(C)C)C=2)C=C2C=1C=C(N(C)C)C=C2

INCIMLINXXICKS-UHFFFAOYSA-M

InChI=1S/C17H19N2O.ClH/c1-18(2)14-7-5-12-9-13-6-8-15(19(3)4)11-17(13)20-16(12)10-14;/h5-11H,1-4H3;1H/q+1;/p-1

[6-(dimethylamino)xanthen-3-ylidene]-dimethylazanium;chloride

Pyronin Y; Pyronine

2934.99.03.00

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.

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

DMSO : ~25 mg/mL (~82.56 mM)
H2O : ~4 mg/mL (~13.21 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.)