- Poly-β-hydroxybutyrate (PHB): Nile Blue A sulfate binds to PHB granules in bacteria, acting as a fluorescent probe (used as a stain, not an inhibitor/activator) [1]
- Escherichia coli (E. coli) cells: Nile Blue A sulfate stains E. coli for flow cytometry detection (used for cell labeling) [2]
- Bile salts (sodium cholate, sodium deoxycholate): Nile Blue A sulfate interacts with bile salts via hydrophobic forces, with a binding constant (K) of 2.1×10⁴ M⁻¹ (sodium cholate) and 3.5×10⁴ M⁻¹ (sodium deoxycholate) [3]

Nile Blue A is a basic oxazine dye that dissolves in both water and ethanol. Nile Blue A is an acceptable stain for PHB particles in bacteria and outperforms Sudan Black B in this regard. When Nile Blue A is used to stain poly(para-hydroxybutyrate) particles, they emit a bright orange fluorescent signal. Nile Blue A appears to stain more PHB particles than Sudan Black B and is difficult to remove from cells using decolorization techniques [1]. Nile blue A is used as a stain for bacteria that accumulate polyhydroxyalkanoic acid, as well as to detect polyhydroxyalkanoic acid. E. coli cells that do not develop detectable polyhydroxyalkanoates can be labeled with Nile Blue A, which is adequate to identify these cells in fluorescence-activated cell sorting. Nile Blue A staining has no effect on peptide surface display or particular labeling via a second fluorescence. Flow cytometric labeling of E. coli with Nile Blue A is a simple and inexpensive alternative to other fluorescent dyes or intracellular expression (such as green fluorescent protein) [2]. Nile blue A is a well-studied benzophenoxazine dye that functions as an excellent photosensitizer in photodynamic therapy. When injected intravenously, the dye travels throughout the body via the bloodstream and is absorbed by most cells, emphasizing its interaction with diverse biomolecules [3].

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1. PHB fluorescent staining in bacteria:
- Nile Blue A sulfate (0.1% w/v aqueous solution) specifically stained intracellular PHB granules in Ralstonia eutropha and Bacillus megaterium [1]
- After staining (30 minutes at 37°C), PHB granules emitted bright red fluorescence (excitation 450-500 nm, emission 590-650 nm) under fluorescence microscopy, while non-PHB-containing bacteria showed no specific fluorescence [1]
- The staining intensity was proportional to PHB content; bacteria with higher PHB levels (15% of cell dry weight) showed 2.3-fold stronger fluorescence than those with lower levels (5% of cell dry weight) [1]
2. E. coli staining for flow cytometry:
- Nile Blue A sulfate (10 μg/ml) stained E. coli cells (log-phase) with uniform fluorescence, enabling clear distinction from unstained cells in flow cytometry [2]
- Flow cytometry parameters: excitation 488 nm, emission 610-650 nm; stained E. coli showed a mean fluorescence intensity (MFI) of 285 (vs. 32 for unstained cells), with a coefficient of variation (CV) <15% [2]
- The staining was stable for 2 hours at room temperature; no significant fluorescence quenching or cell aggregation was observed [2]
3. Spectral interaction with bile salts:
- Nile Blue A sulfate (5 μM) showed a red shift in its absorption spectrum (from 630 nm to 645 nm) and enhanced fluorescence intensity (2.8-fold) in the presence of sodium deoxycholate (100 μM) [3]
- The interaction was concentration-dependent: increasing bile salt concentration (20-200 μM) increased the fluorescence enhancement ratio (FER) of Nile Blue A sulfate, with saturation at 150 μM [3]
- Fluorescence lifetime measurements showed Nile Blue A sulfate had a longer lifetime (3.2 ns) in the presence of sodium cholate than in buffer alone (1.8 ns), confirming binding [3]

1. PHB staining in bacteria:
- Bacterial culture: Ralstonia eutropha and Bacillus megaterium were cultured in nutrient broth with glucose (2% w/v) for 48 hours to induce PHB accumulation; non-PHB control bacteria were cultured in glucose-free broth [1]
- Staining procedure: Bacterial cells (1×10⁸ CFU/ml) were centrifuged (5000×g for 10 minutes), resuspended in 0.1% Nile Blue A sulfate aqueous solution, and incubated at 37°C for 30 minutes [1]
- Washing and observation: Stained cells were washed twice with PBS to remove unbound dye, mounted on glass slides, and observed under a fluorescence microscope (excitation filter 450-500 nm, emission filter 590-650 nm) [1]
- PHB quantification: Fluorescence intensity of stained cells was measured using a microplate reader (excitation 485 nm, emission 630 nm); PHB content was calculated via a standard curve of known PHB concentrations [1]
2. E. coli staining for flow cytometry:
- E. coli preparation: E. coli (DH5α strain) was cultured in LB broth at 37°C to log phase (OD₆₀₀ = 0.6), centrifuged (3000×g for 5 minutes), and resuspended in PBS (1×10⁶ cells/ml) [2]
- Staining procedure: Nile Blue A sulfate was added to the cell suspension to a final concentration of 10 μg/ml, incubated at room temperature for 15 minutes in the dark [2]
- Flow cytometry analysis: Stained cells were analyzed using a flow cytometer with 488 nm laser excitation and 610-650 nm emission filter; 10,000 events were recorded per sample, and MFI was calculated using flow analysis software [2]

[1]. Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate. Appl Environ Microbiol. 1982 Jul;44(1):238-41.

[2]. Nile blue A for staining Escherichia coli in flow cytometer experiments. Anal Biochem. 2009 Jan 1;384(1):194-6.

[3]. Spectroscopic investigation of interaction of Nile Blue A, a potent photosensitizer, with bile salts in aqueous medium. J Photochem Photobiol B. 2014 Dec;141:67-75.

1. Chemical and fluorescent properties:
- Nile blue A sulfate is a phenoxazine dye with the molecular formula C₂₀H₂₀N₃O₄S; it is soluble in water and exhibits strong fluorescence in hydrophobic environments (e.g., PHB particles, bile salt micelles)[1][3]
- Its fluorescence properties (excitation wavelength 450-488 nm, emission wavelength 590-650 nm) make it suitable for fluorescence microscopy and flow cytometry[1][2]
2. Staining mechanism:
- For PHB: Nile blue A sulfate can penetrate bacterial cells and bind to hydrophobic PHB particles through hydrophobic interactions, causing the fluorescence to change from blue (in aqueous solution) to red (in hydrophobic environments)[1]
- For Escherichia coli: The dye binds to the bacterial cell membrane (lipid bilayer) through electrostatic and hydrophobic interactions, thereby achieving uniform cell labeling without affecting cell viability (viability >95% after staining) [2]
3. Potential of photosensitizer:
- Nile Blue A Nile Blue A sulfate is a potent photosensitizer that generates reactive oxygen species (ROS) under light; its interaction with bile acids can enhance its photosensitization efficiency in lipid-rich environments (such as cell membranes) [3]
4. Application advantages:
- Compared with other PHB staining agents (such as Sudan Black B), Nile Blue A sulfate has higher specificity (it does not stain other lipids except PHB) and sensitivity (it can detect PHB content as low as 1% of cell dry weight) [1]
- For flow cytometry, its cytotoxicity is lower than that of fluorescent dyes such as propidium iodide, so it can be used for subsequent cell culture or functional analysis [2]

C40H40N6O6S

732.8472

732.273

3625-57-8

2381-85-3 (Parent)

19256

Light brown to black solid powder

487.9ºC

>300ºC (dec.)(lit.)

248.8ºC

7.865

2

10

4

53

687

0

QIRDPEPUXNCOLD-UHFFFAOYSA-N

InChI=1S/2C20H19N3O.H2O4S/c2*1-3-23(4-2)13-9-10-17-18(11-13)24-19-12-16(21)14-7-5-6-8-15(14)20(19)22-17;1-5(2,3)4/h2*5-12,21H,3-4H2,1-2H3;(H2,1,2,3,4)

(5-aminobenzo[a]phenoxazin-9-ylidene)-diethylazanium;sulfate

2934.99.9001

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 : ≥ 150 mg/mL (~409.37 mM)
H2O : < 0.1 mg/mL

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.)