Fluorescent probe/dye

Cell Labeling Protocol (Guideline)
Note: This protocol serves as a recommended guideline and should be adapted based on specific experimental requirements[1].
Procedure:
1. Cell Preparation
o Seed cells in 20 mm confocal dishes at a density of 5 × 10⁴ cells/mL.
o Incubate cells under standard culture conditions.

2. Probe Staining
o For confocal imaging, add 5 μM meso-Benzothiazole-BODIPY 505/515 (Probe 1) to the culture medium.
o Incubate cells with the probe for 30 minutes.

3. Imaging Parameters
o Excitation wavelength: 488 nm
o Emission collection range: 520–600 nm

Immunofluorescence[1]
Cell Types: SH-SY5Y cells
Tested Concentrations: 5 μM
Incubation Duration: 30 min
Experimental Results: demonstrated relatively weak fluorescence emissions in low viscos cells, but demonstrated strong fluorescence emissions when the SH-SY5Y cells were preincubated with LPS and nystatin.

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Cellular Viscosity Imaging and Autophagy Monitoring [1]
The confocal imaging was carried out using 20 mm confocal dishes with SH-SY5Y cells of 5 × 104 cells/mL. For the subcellular imaging, 1 (5 μM) and Mito-tracker Blue or Lyso-tracker Blue (1 μM) in Dulbecco’s modified Eagle’s medium (DMEM) were used to culture the cells for 30 min. After the cells were washed with phosphate-buffered saline (PBS) three times, a Leica TCS SP8 confocal microscope was used to conduct subcellular imaging with a 100 × oil-immersion objective lens. For Mito-tracker Blue or Lyso-tracker Blue, the cells were excited at 405 nm and emissions were collected at the range of 425–500 nm, which was labeled as the blue channel. For 1, the cells were excited at 488 nm and emissions were collected at 520–600 nm, which was labeled as the red channel. The merged blue and red channels were also given.

For the viscosity-responsive confocal imaging, the cells were first cultured with none, lipopolysaccharide (LPS), or nystatin (both at 20 μM) for 40 min. After washing with PBS three times, the freshly prepared stock solutions of 1 (5 μM in DMEM) were added to the above cell plates, which were further cultured at 37 °C for 30 min. The cells were excited at 488 nm and emissions were collected at 520–600 nm with a 63 × oil-immersion objective lens.

For monitoring viscosity changes of 1 in living cells, low temperature and dexamethasone were used. The cells were first treated with 1 (10 μM) at 37 and 4 °C for 30 min, respectively. The other two groups were first incubated with 1 (10 μM) at 37 °C for 30 min, and then treated with dexamethasone (100 μM) or dimethylsulfoxide (DMSO, 10 μL) for another 10 min. After washing three times with PBS, the cells were excited at 488 nm and emissions were collected at 520–600 nm with a 100 × oil-immersion objective lens.

For monitoring the autophagy process through the lysosomal viscosity changes, SH-SY5Y cells were incubated with 1 (10 μM) at 37 °C for 30 min and then were cultured in Hank’s balanced salt solution (HBSS), normal medium, or HBSS with the addition of 3-methyladenine (3-MA) (an autophagy inhibitor) to give the starvation conditions, rich-nutrient conditions, or autophagy-inhibited conditions for 2 h, respectively. The cells were excited at 488 nm and emissions were collected at 520–600 nm with a 100 × oil-immersion objective lens.

[1]. Novel Meso-Benzothiazole-Substituted BODIPY-Based AIE Fluorescent Rotor for Imaging Lysosomal Viscosity and Monitoring Autophagy. Anal Chem. 2022 Oct 25;94(42):14707-14715.

Meta-substituted boron dipyrrole methylene (BODIPY) offers a potential and innovative strategy for the synergistic construction of aggregation-induced emission (AIE) probes and fluorescent rotors for monitoring changes in cell viscosity, which is crucial for understanding the role of viscosity in closely related diseases. Therefore, we have for the first time rationally designed and synthesized a BODIPY-based fluorescent probe (1) with a rotatable meta-benzothiazole group, exhibiting good viscosity responsiveness and AIE properties. Probe 1, through direct attachment to the thiazole group, shows almost no emission in low-viscosity solvents; however, a strong emission peak appears at 534 nm, gradually increasing with increasing viscosity, attributed to the effective confinement of the rotatable meta-benzothiazole group. In methanol/glycerol mixtures, with viscosities ranging from 0.59 to 945 cP, the fluorescence intensity (log I534) exhibits a good linear relationship with viscosity (log η). Interestingly, compound 1 shows a higher emission intensity at 534 nm in a 70% aqueous solution than in pure acetonitrile solution, likely due to aggregation-induced rotational inhibition. Cell imaging showed that compound 1 was able to successfully sense changes in lysosomal viscosity induced by lipopolysaccharide, nystatin, low temperature and dexamethasone in living cells, which could be further applied to monitor autophagy by tracking changes in viscosity. In contrast, its analog 2, which is directly linked to a phenyl group, did not show viscosity response or aggregation-induced emission (AIE) properties. Therefore, we report for the first time a fluorescent rotor based on mesobenzothiazole-BODIPY that has aggregation-induced emission (AIE) and lysosomal viscosity response properties in nerve cells and can be further applied to autophagy monitoring. This work provides an innovative strategy for designing potential AIE and viscosity-responsive probes. [1]

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In summary, we introduce a rotatable lysosomal-targeting benzothiazole group into the BODIPY core for the first time, and report an AIE fluorescent probe based on mesobenzothiazole BODIPY that can use the free rotation of the mesobenzothiazole group to image lysosomal viscosity in living cells and monitor autophagy. By directly attaching the mesobenzothiazole group to the thiazole moiety, probe 1 exhibited a redshift in both absorption and emission spectra compared to probe 2, which was directly attached via a phenyl group. More interestingly, even with slightly different attachment methods, probe 1 and probe 2 displayed distinctly different properties, such as viscosity responsiveness and aggregation-induced emission (AIE) characteristics. At low viscosity, probe 1 showed almost no fluorescence emission; as viscosity gradually increased, a strong fluorescence emission band appeared at 534 nm. Notably, this meta-benzothiazole-substituted probe exhibited excellent AIE characteristics at 534 nm, further confirming that the rotation of the meta-benzothiazole group can be effectively restricted in both aggregated states and high viscosity environments. Further cell experiments showed that the meta-benzothiazole group can also serve as a lysosomal targeting group, and its application in cell viscosity monitoring was successful after pretreatment with LPS, nystatin, low temperature, and dexamethasone. Furthermore, the viscosity-responsive properties of probe 1 were also applied to autophagy monitoring. In summary, we first used the uncommon meta-phenyl substitution to introduce the meta-benzothiazole group into the core of BODIPY by direct connection with a five-membered ring, studied its viscosity/aggregation-induced emission (AIE) properties, and further applied it to the visualization of cell viscosity changes and the monitoring of lysosomal autophagy. Other related studies on BODIPY based on meta-five-membered heterocyclic substitution are underway, aiming to optimize its AIE/viscosity properties and study its structure-property relationship. [1]

C20H18BF2N3S

381.25

Typically exists as solid at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)