Fluorescent Dye; NAD (P) H-oxidoreductases
1. Preparation of MTT working solution:
MTT was dissolved with PBS to obtain 5 mg/mL of MTT.
2. Cell proliferation test (96-well plate).
2.1 Inoculated cells: The prepared single cells were prepared with a suspension containing 10%FBS and cultured a 96-well plate with 1000-10000 cells per well and a volume of 100μL per well.
2.2 Cultured cells: 37℃. 5% CO2, incubated for 24-72 h.
2.3 Add 10 μL MTT to each well, incubate for 4 h, discard the supernatant (centrifuge first if using suspended cells).
2.4 Add 100 μL DMSO and shake for 10 minutes to completely dissolve the crystal.
2.5 Monitor the increase in absorbance at OD=562 nm with an absorbance board reader.
MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide) is a monotetrazolium salt that acts as an electron acceptor. It is reduced by various cellular oxidoreductase enzymes, primarily utilizing the reduced pyridine nucleotide cofactor NADH. [1]
It is also reduced by succinate dehydrogenase (mitochondrial Complex II) and can be reduced by superoxide. [1]

1. Preparation of MTT working solution:
MTT was dissolved with PBS to obtain 5 mg/mL of MTT.
2. Cell proliferation test (96-well plate).
2.1 Inoculated cells: The prepared single cells were prepared with a suspension containing 10%FBS and cultured a 96-well plate with 1000-10000 cells per well and a volume of 100μL per well.
2.2 Cultured cells: 37℃. 5% CO2, incubated for 24-72 h.
2.3 Add 10 μL MTT to each well, incubate for 4 h, discard the supernatant (centrifuge first if using suspended cells).
2.4 Add 100 μL DMSO and shake for 10 minutes to completely dissolve the crystal.
2.5 Monitor the increase in absorbance at OD=562 nm with an absorbance board reader.

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MTT reduction is widely used as a colorimetric assay to measure the metabolic activity and proliferation of viable cells in culture. The reduction signal correlates with the integrated pyridine nucleotide redox status of cells. [1]
Subcellular fractionation studies indicate that NADH is the most favored substrate for MTT reduction, while succinate accounts for less than 10% of the total reducing potential in cell homogenates. [1]
In vitro, MTT can be reduced by NADH, NADPH, or succinate in the presence of crude cell fractions, with NADH being the most efficient electron donor. [1]

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MTT reduction is widely used as a colorimetric assay to measure the metabolic activity and proliferation of viable cells in culture. The reduction signal correlates with the integrated pyridine nucleotide redox status of cells. [1]
Subcellular fractionation studies indicate that NADH is the most favored substrate for MTT reduction, while succinate accounts for less than 10% of the total reducing potential in cell homogenates. [1]
In vitro, MTT can be reduced by NADH, NADPH, or succinate in the presence of crude cell fractions, with NADH being the most efficient electron donor. [1]

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.[1]

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The capacity for MTT reduction is widely distributed in various subcellular fractions (nuclear, mitochondrial, microsomal, cytosolic) when NADH is used as a substrate. [1]
In vitro assays with optimum substrate concentrations showed that NADH is the most favored substrate for MTT reduction, while succinate is the least favored. [1]
The succinate-dependent MTT reduction activity is sensitive to the Complex II inhibitor thenoyltrifluoroacetone (TTFA), confirming mitochondrial Complex II as one site of reduction. [1]

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The capacity for MTT reduction is widely distributed in various subcellular fractions (nuclear, mitochondrial, microsomal, cytosolic) when NADH is used as a substrate. [1]
In vitro assays with optimum substrate concentrations showed that NADH is the most favored substrate for MTT reduction, while succinate is the least favored. [1]
The succinate-dependent MTT reduction activity is sensitive to the Complex II inhibitor thenoyltrifluoroacetone (TTFA), confirming mitochondrial Complex II as one site of reduction. [1]

The MTT assay is simple, accurate and yields reproducible results. This method has been developed originally by Mossman in 1983. The key component is (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide) or MTT. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material (IC50).

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A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision.
PMID:6606682

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/The authors/ compared the MTT assay to the standard clonogenic assay and had good agreement of surviving fraction /of murine solid tumor cells/ after radiation doses of 2 and 4 Gy. It is possible, therefore, to adapt the MTT assay for use with cell suspensions prepared directly from fresh murine tumors. This may provide a methodology for the determination of the clinical radiosensitivity of tumors including fresh clinical tumor specimens.
PMID:3417490

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A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtiter plate spectrophotometer.
https://pubchem.ncbi.nlm.nih.gov/compound/64965

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Cell Proliferation/Cytotoxicity Assay: The standard microplate assay involves incubating cells with MTT (typically at a concentration of 500 µg/ml) for a period (e.g., 1-4 hours). Metabolically active cells reduce the yellow MTT to purple, insoluble formazan crystals. The formazan product is then solubilized (e.g., using DMSO or acidified isopropanol) and the absorbance is measured spectrophotometrically, usually at 570 nm. The absorbance is proportional to the number of viable, metabolically active cells. [1]
Subcellular Localization Studies: Confocal imaging studies with HepG2 cells indicated that most MTT-formazan deposits are not coincident with mitochondria but occur in the cytoplasm and in proximity to the plasma membrane. [1]
Mechanistic Studies: Treatment of cells with inhibitors of glucose transport (2-deoxyglucose) and glycolysis (iodoacetamide) strongly inhibits MTT reduction. In contrast, the succinate dehydrogenase inhibitor TTFA has no effect, and inhibitors of mitochondrial electron transport (cyanide, azide, rotenone) can stimulate or have little effect on MTT reduction in the short term. [1]
Superoxide Involvement: Using SOD and inhibitors, it was demonstrated that 20-30% of MTT reduction inside HeLa cells could be attributed to superoxide, while 80% of extracellular reduction was SOD-sensitive. [1]

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Cell Proliferation/Cytotoxicity Assay: The standard microplate assay involves incubating cells with MTT (typically at a concentration of 500 µg/ml) for a period (e.g., 1-4 hours). Metabolically active cells reduce the yellow MTT to purple, insoluble formazan crystals. The formazan product is then solubilized (e.g., using DMSO or acidified isopropanol) and the absorbance is measured spectrophotometrically, usually at 570 nm. The absorbance is proportional to the number of viable, metabolically active cells. [1]
Subcellular Localization Studies: Confocal imaging studies with HepG2 cells indicated that most MTT-formazan deposits are not coincident with mitochondria but occur in the cytoplasm and in proximity to the plasma membrane. [1]
Mechanistic Studies: Treatment of cells with inhibitors of glucose transport (2-deoxyglucose) and glycolysis (iodoacetamide) strongly inhibits MTT reduction. In contrast, the succinate dehydrogenase inhibitor TTFA has no effect, and inhibitors of mitochondrial electron transport (cyanide, azide, rotenone) can stimulate or have little effect on MTT reduction in the short term. [1]
Superoxide Involvement: Using SOD and inhibitors, it was demonstrated that 20-30% of MTT reduction inside HeLa cells could be attributed to superoxide, while 80% of extracellular reduction was SOD-sensitive. [1]

Toxicological Information
Interactions
KB cells were treated with MTT at final concentrations up to 0.2 mg/ml, alone or in combination with methotrexate (10 and 32 μM). When used alone, MTT at concentrations as low as 0.025 mg/ml extended the population doubling time from 26 hours (control group) to 96 hours. At concentrations of 0.2 mg/ml or higher, the total cell count decreased. Treatment with 0.1 mg/ml MTT for 8 hours resulted in a reduction in the number of cells in S and G2/M phases. However, no significant effects were observed at 2 and 4 hours. The effect of 0.2 mg/ml MTT was more rapid and significant, leading to accumulation of cells in G0/G1 and S phases. MTT in combination with methotrexate more significantly disrupted the KB cell cycle.
…Phenyrazine methyl ester (PMS) and MTT are often used in combination: however, PMS did not enhance the mutagenicity of MTT. Venitt S, Crofton-Sleigh; Mutation Research/Genetic Toxicology 68 (2): 107-116 (1979)

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Antidote and First Aid Treatment
Basic Treatment: Maintain an open airway. Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat if necessary… Monitor for shock and treat if necessary… Anticipate seizures and treat if necessary… If eyes are contaminated, flush them immediately with water. Continuously flush eyes with saline solution during transport… Do not use emetics. If swallowed, rinse mouth and dilute with 5 ml/kg to 200 ml of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After disinfecting skin burns, cover with a dry, sterile dressing… /Class A and B Poisoning/
Advanced Treatment: For patients with impaired consciousness, severe pulmonary edema, or respiratory arrest, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Monitor heart rhythm and treat arrhythmias as needed…. Establish intravenous access using 5% glucose solution (SRP: “Keep it patent,” minimum flow rate). If signs of hypovolemia appear, use lactated Ringer's solution. Watch for signs of fluid overload. Consider medical treatment for pulmonary edema…. Administer fluids with caution in cases of hypotension with signs of hypovolemia. Watch for signs of fluid overdose…. Treat seizures with diazepam (Valium)…. Use promecaine hydrochloride as an adjunct to eye irrigation… /Toxins A and B/

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Non-human Toxicity Excerpt
/Genotoxicity/The combination of tetrazolium salts with phenazine methyl ester sulfate (PMS) is a widely used laboratory reagent. PMS and three tetrazolium salts (MTT, [3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazol bromide]; TTC, [2,3,5-triphenyltetrazol chloride]; and NBT, [2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4')]) were tested for mutagenicity in various Escherichia coli WP2 (trp-) and Salmonella typhimurium strains. In the absence of S-9, PMS was mutagenic to Escherichia coli uvrApKM101 and Salmonella typhimurium TA 100 and TA 98, with linear dose-response curves in the dose range of 0.5–10 μg/plate, with slopes of 2.3, 1.3, and 0.5 revertant mutants/nmol, respectively. The addition of S-9 significantly reduced the mutagenicity of PMS in these bacteria. In the absence of S-9, MTT was mutagenic to E. coli uvrApKM101, Salmonella typhimurium TA 100, and TA 98, with linear dose-response curves at doses ranging from 0.5 to 10 or 50 μg/plate, with slopes of 3.8, 12.1, and 1.0, respectively. The addition of S-9 significantly reduced the mutagenicity of MTT. MTT showed weak mutagenicity against E. coli WP2 (0.22 revertant mutants/nmol), significantly enhanced mutagenicity against WP2 uvrA (1.29 revertant mutants/nmol), and no mutagenicity against WP2 lexA (due to a negative slope from toxicity), suggesting that MTT causes excisable misrepair DNA damage. When PMS and MTT are often used in combination: however, PMS did not enhance the mutagenicity of MTT.
/Alternative In Vitro Assays/ ... /Authors/ ... The effects of MTT on cell growth and cell cycle distribution were investigated using DNA flow cytometry. KB cells were treated with MTT at final concentrations up to 0.2 mg/ml, either alone or in combination with methotrexate (10 and 32 μM). When used alone, MTT at concentrations as low as 0.025 mg/ml extended the population doubling time from 26 hours (control group) to 96 hours. At concentrations of 0.2 mg/ml or higher, the total cell count decreased. After 8 hours of treatment with 0.1 mg/ml MTT, the number of cells in S and G2/M phases decreased. However, no significant effects were observed at 2 and 4 hours. The effect of 0.2 mg/ml MTT was more rapid and significant, leading to cell accumulation in G0/G1 and S phases. When MTT was used in combination with methotrexate, the KB cell cycle was more significantly perturbed. Although the MTT assay measures cell viability and proliferation via mitochondrial dehydrogenases, it also showed a significant effect on cellular DNA content and cell proliferation. These observations question the reliability of such an assay, suggesting that it may measure the combined effect of MTT and the drug, rather than the effect of the drug itself.
/Other Toxicity Information/ Although various detection methods for tetrazolium compounds are widely used, the bioreduction mechanisms of these compounds are not fully elucidated. /Authors/ The ability of tetrazolium salts to penetrate intact cell membranes was investigated. 5-Cyano-2,3-dimethyltetrazole chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) tetrazolium salts appear to be examples of substances reduced through different mechanisms. /Authors/ Evidence provided suggests that MTT readily crosses the cell membrane. Intact cell membranes are able to penetrate tetrazolium salts and be reduced intracellularly. MTT appears to be reduced by both cell membrane reductases and intracellular reductases; the reduced cells are not damaged and retain metabolic activity for at least 45 minutes. In contrast, CTC remains extracellular relative to living cells and therefore requires cell membrane-permeable electron carriers for efficient reduction. However, reduction of CTC in the presence of electron carriers damages the cell membrane. The reduction sites (intracellular and extracellular) of tetrazolium salts are determined based on the deposition of formazan. Formazan crystals are detected using fluorescence or backscattered light confocal laser microscopy. The authors speculate that the ability of tetrazolium salts to cross the intact plasma membrane is an important experimental variable that needs to be controlled in order to correctly interpret the results of tetrazolium salt assays intended to measure cellular oxygen free radical generation, mitochondrial, cytoplasmic, or outer membrane reductase activity. PMID:10900139

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Large, insoluble molecules formed intracellularly, such as MTT-formazan crystals, can be expelled via exocytosis. This process may lead to cell death, possibly due to the crystals penetrating the cell membrane. [1]

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Large, insoluble MTT-formazan crystals formed intracellularly can be expelled via exocytosis. This process may lead to cell death, possibly due to the crystals penetrating the cell membrane. [1]

[1]. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11:127-52.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide is the bromide salt of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol. It is used as a dye and colorimetric reagent. It contains 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol.

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Source/Uses
For bioassays;
Tetrazol salts are used to detect dehydrogenase activity or any other enzyme system that produces redox equivalents. Due to this property, they are very useful in academic and clinical research and many diagnostic applications, such as: (1) AIDS and cancer research, cell and molecular biology: for detecting cell proliferation and cell viability and cytotoxicity assays; (2) enzyme diagnostics and clinical chemistry: for determining dehydrogenase activity and other enzyme systems that form redox equivalents; for determining dehydrogenase substrate concentrations; for detecting glucose, ethanol, glycerol and other substrates that produce redox equivalents in coupling reactions; (3) immunohistochemistry: for detecting alkaline phosphatase; (4) histology and pathology: for histochemical detection of dehydrogenases; (5) in seed culture: for testing seed viability.
SERVA Tetrazolium Salts - Datasheet. Available as of March 28, 2005 at: https://www.serva.de/products/latest/tetrazolium.shtml
Tetrazolium salts are widely used to detect the redox potential of cells for cell viability, cytotoxicity and proliferation assays. …The MTT reduction method remains the most commonly used method for tetrazolium salt-based cell viability assays.
Tetraazole salt used to detect redox potential in living cells and tissues: 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide. Applications: superoxide produced by fumarate reductase and nitric oxide synthase; mitochondrial dehydrogenase activity; cell viability and proliferation; neuronal cell death; platelet activation; tumor cell adhesion and invasion; multidrug resistance; in vitro toxicity test /Excerpt from table/
The MTT assay is commonly used for screening anti-HIV and anti-tumor drugs to assess cell viability and proliferation.

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MTT is one of the most commonly used tetraazole salts in cell biology for measuring cellular metabolic activity, proliferation and cytotoxicity. [1]
It is a positively charged lipophilic monotetraazole salt that can easily enter living cells via plasma membrane potential. [1]
Its reduction mainly occurs intracellularly and is associated with a variety of NADH-utilizing redox enzymes, not just mitochondrial succinate dehydrogenase. Reduction sites include the cytoplasm, non-mitochondrial membranes (endosomes/lysosomes, plasma membranes), and mitochondria. [1]
The generated formazan product is insoluble in water, so a dissolution step is required before spectrophotometer readings, making it an endpoint detection method. [1]
It has been used in drug screening assays (e.g., the National Cancer Institute) and to predict chemotherapeutic sensitivity and resistance to cancer drugs. [1]
It is also used to measure superoxide produced by activated neutrophils, a large portion of which is reduced extracellularly and is sensitive to superoxide dismutase (SOD). [1]
Limitations include the assumption that dye reduction is proportional to cell number, which is affected by growth conditions, cell cycle stage, and cellular metabolic state. [1]

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MTT is one of the most widely used tetrazolium salts in cell biology for measuring cellular metabolic activity, proliferation, and cytotoxicity. [1]
It is a positively charged, lipophilic monotetrazolium salt that can easily enter living cells via plasma membrane potential. [1]
Its reduction mainly occurs intracellularly and is associated with a variety of NADH-utilizing oxidoreductases, not just mitochondrial succinate dehydrogenase. Reduction sites include the cytoplasm, non-mitochondrial membranes (endosomes/lysosomes, plasma membrane), and mitochondria. [1]
The resulting formazan product is insoluble in water, so a dissolution step is required before spectrophotometer readings, making it an endpoint detection method. [1]
It has been used in drug screening tests (e.g., the National Cancer Institute) and in predicting chemotherapeutic sensitivity and resistance to cancer drugs. [1]
It has also been used to measure superoxide produced by activated neutrophils, a significant portion of which is reduced extracellularly and is sensitive to superoxide dismutase (SOD). [1]
Its limitations include the assumption that dye reduction is proportional to cell number, which is affected by growth conditions, cell cycle stage, and cellular metabolic state. [1]

C18H16BRN5S

414.32

413.03

C, 52.18; H, 3.89; Br, 19.29; N, 16.90; S, 7.74

298-93-1

13146-93-5 (parent)

64965

Light yellow to yellow solid powder

195 °C (dec.)(lit.)

0.288

0

5

3

25

410

0

AZKSAVLVSZKNRD-UHFFFAOYSA-M

InChI=1S/C18H16N5S.BrH/c1-13-14(2)24-18(19-13)23-21-17(15-9-5-3-6-10-15)20-22(23)16-11-7-4-8-12-16;/h3-12H,1-2H3;1H/q+1;/p-1

2-(3,5-diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole;bromide

Thiazolyl Blue; Thiazolyl Blue Tetrazolium Bromide; 298-93-1; MTT; MMT Tetrazolium; Methylthiazoletetrazolium; Thiazolyl Blue Monotetrazolium; 2348-71-2; Methylthiazolyldiphenyl-tetrazolium bromide

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 : ~25 mg/mL (~60.34 mM)
H2O : ~0.88 mg/mL (~2.12 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: 6.67 mg/mL (16.10 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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