- Amyloid fibrils – Binds to amyloid fibrils via micelle formation, leading to enhanced fluorescence emission [1]
- Nucleic acids (DNA/RNA) – Binds to negatively charged nucleic acids, likely through electrostatic interactions between positively charged ThT micelles and negatively charged nucleic acids [1]

- Thioflavin T exhibits enhanced fluorescence upon binding to amyloid fibrils; a new excitation peak appears at 450 nm upon binding, responsible for enhanced fluorescence emission at 482 nm [1]
- In aqueous solutions, Thioflavin T forms micelles at concentrations commonly used to monitor fibrils by fluorescence assay (~10-20 μM); the critical micellar concentration was calculated to be 4.0 ± 0.5 μM by conductivity measurements [1]
- Fluorescence excitation and emission properties of Thioflavin T are dependent on micelle formation; significant increase in excitation and emission intensity was observed above 5 μM concentration [1]
- Fluorescence anisotropy of Thioflavin T increased starting at 0.5 μM and reached maximum at 20 μM, indicating molecular association [1]
- Thioflavin T binds to inclusion bodies of interleukin-2, showing 50-100 fold enhancement of fluorescence; however, other protein aggregates such as aggregated P22 tail-spike protein, heat-induced protein aggregates from HeLa cells, and amorphous aggregates of SMA VL did not result in enhanced Thioflavin T fluorescence [1]
- Amyloid fibrils derived from various proteins (A-β peptide, insulin, transthyretin, α-synuclein, amylin, lysozyme, Ig light chains) significantly enhance Thioflavin T fluorescence upon binding [1]
- A 13-amino acid peptide ED (EDVAVYYCHQYYS) that forms fibrils showed binding of Thioflavin T by enhanced fluorescence and micelles bound along fibril length; another peptide KLEG (KLKLKLELELELELG) with positive charges (three lysine residues) showed no enhanced Thioflavin T fluorescence, suggesting that positive charges repel positively charged ThT micelles [1]
- Thioflavin T binding to amyloid fibrils is reduced several-fold when pH is decreased below 3 [1]
- Increasing salt concentration from 0.5 to 2 M NaCl caused a small decrease (30%) in fluorescence emission of Thioflavin T bound to amyloid fibrils, indicating that salt does not remove bound ThT micelles [1]

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Thioflavin T (ThT) is a benzothiazole dye that is frequently used to detect amyloid fibrils both in vitro and ex vivo. It exhibits heightened fluorescence upon binding to amyloid fibrils. Thioflavin T is present in aqueous solutions as micelles at concentrations of around 10–20 μM, which are frequently employed to monitor fibrils using fluorimetry. After measuring specific conductivity changes at various thioflavin T concentrations, 4.0±0.5 μM was determined to be the crucial micelle concentration. The generation of micelles affects thioflavin T's fluorescence excitation and emission as well. Using atomic force microscopy, thioflavin T micelles with a diameter of 3 nm were directly detected, and along the fiber length, thioflavin T micelle production with unique fibrils was noted. An increase in the number of micelles bound along the fiber length is shown by rising thioflavin T concentrations above the critical micelle concentration. Atomic force microscopy shows that thioflavin T micelles are broken down at low pH levels and that the fluorescence increase that results from thioflavin T binding to amyloid fibrils also declines many times below 3. Amyloid fibrils are bound by thioflavin T micelles, which increases fluorescence emission [1].

An animal study showed that in a high-fat diet-induced obese mouse model, ThT treatment modulated serum levels of adipokine hormones (such as adiponectin and leptin), reduced insulin and HOMA-IR levels, indicating potential ameliorative effects on metabolic disorders. ThT is also used for histochemical staining of amyloid plaques in animal tissues.

A typical ThT fluorescence binding assay is performed in a buffer (e.g., PBS or Tris-HCl, pH 7.4). A specific concentration of amyloid fibrils or G-quadruplex DNA is mixed with ThT (typically 10-50 µM) and incubated at room temperature (e.g., 10-30 minutes). Fluorescence intensity is read using a microplate reader at excitation 440 nm and emission 480-490 nm. In competition binding assays, ThT is first bound to the target, followed by the addition of test compounds to observe fluorescence quenching.

In cellular assays, ThT is used to detect intracellular protein aggregates. Cells seeded in 96-well plates or dishes are treated or transfected, then the medium is removed and cells are washed with PBS. Cells are incubated with ThT solution (e.g., 10-20 µM in PBS) for 30-60 minutes in the dark. After staining, the ThT solution is removed, cells are washed with PBS, and analyzed by fluorescence microscopy or flow cytometry. The fluorescence intensity is generally positively correlated with the aggregate content within the cells.

In animal models, ThT can be administered via intraperitoneal injection or oral gavage. In one mouse study, ThT (5, 10, 15 mg/kg) was administered by gavage once daily for 4 weeks to evaluate its effects on obesity. For imaging studies, animals can be used for in vivo imaging after intravenous injection of ThT to track amyloid plaques, or they are sacrificed to harvest tissues (e.g., brain or liver) for sectioning and ThT staining.

ThT is a cationic salt (typically chloride) with moderate water solubility. Regarding pharmacokinetics, it readily crosses the blood-brain barrier (BBB), making it an effective probe for studying CNS amyloidosis. Specific half-life and tissue distribution parameters vary by species and administration route, but rapid plasma clearance and tissue distribution have been reported.

According to the Safety Data Sheet, ThT is classified as a hazardous substance. It is toxic if swallowed (H301), causes serious eye damage (H318), may cause an allergic skin reaction (H317), and is very toxic to aquatic life with long-lasting effects (H410). Appropriate protective equipment (gloves, eye protection) should be worn when handling.

[1]. Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol. 2005 Sep;151(3):229-38.

- Thioflavin T is a cationic benzothiazole dye (chemical structure: a hydrophobic end with a dimethylamino group attached to a phenyl group, linked to a polar benzothiazole group containing polar N and S) [1]
- Thioflavin T was introduced by Vassar and Culling in 1959 for detection of amyloid in tissue sections using fluorescence microscopy [1]
- The dye shows enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro [1]
- Thioflavin T also binds to other connective tissues such as cartilage matrix, elastic fibers, and mucopolysaccharides, as well as to DNA and RNA [1]
- Kelenyi (1967) modified staining conditions to lower pH (between 0.8 and 2.8) to improve specificity for amyloid and reduce staining of nucleic acids [1]
- Thioflavin T forms micelles of 3 nm diameter in aqueous solutions, visualized by atomic force microscopy; these micelles bind along the length of amyloid fibrils [1]
- At pH below 3, Thioflavin T micelles are disrupted as observed by AFM, and fluorescence enhancement upon binding to amyloid fibrils is reduced several-fold, suggesting that positive charge on ThT molecule has a role in its micelle formation [1]
- The critical micellar concentration of Thioflavin T in water is 4.0 ± 0.5 μM; in TBS (50 mM Tris, 150 mM NaCl, pH 7.5), micelle size varies from 3 to 6 nm in diameter [1]
- In contrast to Thioflavin T, Congo red did not show characteristic micelles bound to ED fibrils; instead, an increase in fibril heights to 10 ± 0.5 nm was observed, possibly due to Congo red-induced lateral aggregation [1]
- Thioflavin T binding to nucleic acids is considerably reduced at low pH, indicating a role of charged interactions in binding [1]
- Thioflavin T is not specific for β-sheet structure as it binds both nucleic acids and amyloid fibrils; binding to nucleic acids is purely based on charged interactions [1]

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Thioflavin T is an organochloride salt with the counterion 2-[4-(dimethylamino)phenyl]-3,6-dimethyl-1,3-benzothiazol-3-onium. It is widely used for the colorimetric and quantitative analysis of amyloid proteins in vitro and in vivo. It can also be used as a fluorescent dye, histological dye, and anti-aging agent. It contains the thioflavin T cation.

C17H19CLN2S

318.8642

318.095

C, 64.04; H, 6.01; Cl, 11.12; N, 8.79; S, 10.05

2390-54-7

16953

Yellow to orange solid powder

212ºC

0.771

0

3

2

21

325

0

CC1=CC2=C(C=C1)[N+](=C(S2)C3=CC=C(C=C3)N(C)C)C.[Cl-]

JADVWWSKYZXRGX-UHFFFAOYSA-M

InChI=1S/C17H19N2S.ClH/c1-12-5-10-15-16(11-12)20-17(19(15)4)13-6-8-14(9-7-13)18(2)3;/h5-11H,1-4H3;1H/q+1;/p-1

4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline;chloride

Thioflavin T; 2390-54-7; Thioflavine T; Basic yellow 1; Acronol Yellow TC;

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 : ~16.67 mg/mL (~52.28 mM)
H2O : ~5 mg/mL (~15.68 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (5.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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