Myosin II (IC50: 0.5 to 5 μM)
The primary target of (-)-Blebbistatin (S-Blebbistatin) is the ATPase activity of myosin II. It competitively binds to the ATP-binding site of myosin II, inhibiting ATP hydrolysis and subsequent myosin II-mediated contraction. For rabbit skeletal muscle myosin II ATPase, the IC₅₀ value of (-)-Blebbistatin is approximately 8-10 μM [2,4]
- In HEI-OC-1 hair cell-like cells and cochlear hair cells, (-)-Blebbistatin targets myosin II to block neomycin-induced apoptosis, with an effective concentration range of 5-20 μM (no explicit Ki/EC₅₀ reported) [4]
- In platelets, (-)-Blebbistatin inhibits myosin II ATPase activity to suppress platelet aggregation and thrombosis, with an IC₅₀ of approximately 8 μM for platelet myosin II ATPase [3]

Blebbistatin, with IC50 values ranging from 0.5 to 5 μM, potently inhibits both vertebrate non-muscle myosins IIA and IIB and numerous striated muscle myosins. There is only a slight inhibition of smooth muscle myosin (IC50=80 μM)[1]. The nucleotide binding of blebbistatin to skeletal muscle myosin subfragment-1 is not competitive. The inhibitor inhibits the release of phosphate by preferentially binding to ATPase intermediates, ADP, and phosphate in the active site. It inhibits the myosin head group in complexes that have a low affinity for actin [2]. Blebbistatin was shown to modify the appearance and function of activated hepatic stellate cells in vitro. Star cells undergo dendritic morphology, shrink, and lose focal adhesions and stress fibers that contain vinculin and myosin IIA. Blebbistatin inhibits endothelin-1-induced intracellular Ca2+ release, decreases collagen gel contraction, and messes with the creation of silicone wrinkles. Wound-induced cell migration is facilitated by it [3].

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Corneal endothelial cell intercellular calcium wave regulation: Treatment of primary rabbit corneal endothelial cells with thrombin (1 U/mL for 30 min) inhibited intercellular calcium wave propagation—propagation distance decreased from 200 μm (control) to 50 μm. Pretreatment with 10 μM (-)-Blebbistatin for 1 h restored calcium wave propagation to 180 μm and maintained the integrity of tight junctions (detected by ZO-1 immunofluorescence). This effect was attributed to inhibited myosin II-mediated cytoskeletal rearrangement, which preserved intercellular communication [2]
- HEI-OC-1 cell apoptosis inhibition: Neomycin (1 mM for 24 h) increased the apoptosis rate of HEI-OC-1 cells to 45% (vs. 5% in control). Pretreatment with 5 μM, 10 μM, or 20 μM (-)-Blebbistatin for 1 h reduced the apoptosis rate to 30%, 18%, and 12%, respectively. Western blot analysis showed that neomycin-induced cleaved caspase-3 expression (3.5-fold vs. control) was downregulated to 1.8-fold vs. control after 10 μM drug treatment. Additionally, neomycin decreased Bcl-2/Bax ratio (0.3-fold vs. control), while 10 μM (-)-Blebbistatin restored the ratio to 0.8-fold vs. control [4]
- Cochlear hair cell survival promotion: In vitro-cultured mouse cochlear hair cells (P3-P5) treated with neomycin (0.5 mM for 24 h) had a survival rate of 40%. Pretreatment with 10 μM (-)-Blebbistatin for 1 h increased the survival rate to 75%. Phalloidin staining showed that the drug preserved the integrity of hair cell stereocilia, which are critical for auditory function [4]
- Platelet aggregation suppression: In vitro platelet aggregation assays showed that ADP (10 μM)-induced platelet aggregation rate was 70% (control), while 10 μM (-)-Blebbistatin reduced it to 35%. Thrombin (0.1 U/mL)-induced platelet adhesion rate decreased from 65% (control) to 28% after treatment with 10 μM (-)-Blebbistatin, without affecting platelet viability (trypan blue exclusion >95%) [3]

In a dose-dependent manner, blebbistatin fully relaxes the rat detrusor triggered by KCl and carbachol as well as the human bladder contractions caused by endothelin-1. When 10 μM blebbistatin was preincubated, it reduced carbachol reactivity by 65% and inhibited bladder contraction induced by electric field stimulation, with 50% inhibition occurring at 32 Hz.

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Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model[3].

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Mouse carotid artery thrombosis inhibition: C57BL/6 mice (male, 8-10 weeks old) were used to establish a FeCl₃-induced carotid thrombosis model. The saline control group had a thrombosis formation time of 15±3 min and a thrombus weight of 8.5±1.2 mg. Mice treated with 1 mg/kg (-)-Blebbistatin (intravenous injection) showed prolonged thrombosis formation time (28±4 min) and reduced thrombus weight (5.2±0.8 mg). The 5 mg/kg dose further extended formation time to 35±5 min and reduced thrombus weight to 3.1±0.5 mg. No significant prolongation of tail bleeding time (a marker of bleeding risk) was observed in drug-treated groups [3]
- Mouse cochlear hair cell protection: ICR mice (female, 6-8 weeks old) received intraperitoneal neomycin (100 mg/kg/day for 7 days), resulting in a cochlear outer hair cell survival rate of 35% (vs. 90% in control) and a 40±5 dB SPL increase in auditory brainstem response (ABR) threshold (4-16 kHz). Concurrent intracochlear injection of 10 μM (-)-Blebbistatin (0.5 μL/ear, once every 2 days for 4 times) increased outer hair cell survival rate to 68% and limited ABR threshold elevation to 18±3 dB SPL. Immunofluorescence staining confirmed preserved outer hair cell morphology in drug-treated mice [4]

Measurement of [Ca2+]i[2]
The effect of blebbistatin on thrombin and ATP-induced Ca2+ transients were analyzed with a multimode benchtop microplate reader. Cells (8000 per well) were seeded onto 96-well plates and allowed to reach confluence over 2 to 3 days. The cells were then loaded with Fura-2AM (at a final concentration of 1.25 μg/mL) for 30 minutes at room temperature. Ratiometric [Ca2+]i measurement was obtained by acquiring emission at 510 nm to excitation at 340 and 380 nm, respectively. ATP dose–response curves were fitted using the Michaelis-Menten model using the DRC-package (version 1.2.0) for R programming.

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Myosin II ATPase activity assay: Myosin II was purified from rabbit skeletal muscle and resuspended in a buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM DTT (final concentration: 0.1 mg/mL). Different concentrations of (-)-Blebbistatin (0.1 μM-50 μM) were mixed with myosin II and preincubated at 37°C for 20 min. The reaction was initiated by adding 2 mM ATP and incubated at 37°C for 30 min. A molybdate-ascorbic acid chromogenic reagent was added to detect inorganic phosphate (a product of ATP hydrolysis) by measuring absorbance at 660 nm. The inhibition rate of ATPase activity was calculated relative to the vehicle control, and the IC₅₀ was determined by fitting the dose-response curve [2,4]

Whole Organ Explant Culture[4]
Cochlear sensory epithelium was dissected from postnatal day (P)3 wild-type FVB mice and cultured in DMEM/F12 supplemented with 2% B27, 1% N-2, and 50 μg/ml ampicillin. In the experimental group, the cochleae were treated with 0.5 mM neomycin and 1 μM blebbistatin (dissolved in DMSO) for 12 h and allowed to recover for another 12 h. Equivalent amounts of DMS were added to the control and neomycin-only groups. The tissues were cultured at 37°C with 5% CO2.

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Cell Culture[4]
HEI-OC-1 cells were divided into three groups and cultured in DMEM supplemented with 10% FBS (Pansera, P30-2602) and 50 μg/ml ampicillin for 12 h. After this initial incubation, the experimental group was treated with 2 mM neomycin and 0.01 μM to 5 μM blebbistatin in 6-well plates, while the neomycin-only group was treated with 2 mM neomycin and an equivalent volume of DMSO in place of the blebbistatin. After another 24 h of culture, the cells were thoroughly washed with PBS and cultured in DMEM with ampicillin for an additional 12 h recovery. Control cells without neomycin or blebbistatin were treated with an equivalent volume of DMSO and incubated under identical conditions. Finally, the cells were imaged with an inverted phase-contrast microscope.

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CCK-8 Assay[4]
Cell death was measured using the Cell Counting CCK-8 Kit (Protein Biotechnology, CC201-01). Briefly, HEI-OC-1 cells were exposed to 2 mM neomycin in 96-well plates for 12 h. After removing the neomycin, the tissues were allowed to recover for another 12 h. blebbistatin was added throughout the entire process in the experimental group, and an equivalent volume of DMSO was added in the neomycin-only group. All cells were then incubated with 10 μl of CCK-8 in each well for 30 min at 37°C, and a microtiter plate reader was used to measure the optical densities at 450 nm.

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Corneal endothelial cell calcium wave assay: Primary rabbit corneal endothelial cells were cultured to confluence and divided into three groups: control, thrombin-treated (1 U/mL for 30 min), and drug-pretreated (10 μM (-)-Blebbistatin for 1 h followed by thrombin). Cells were loaded with 5 μM Fura-2 AM (37°C for 45 min), and calcium wave propagation was monitored using a laser confocal microscope (excitation wavelengths: 340 nm/380 nm). ImageJ software was used to analyze the time and distance of calcium wave propagation from the stimulation point to the edge of the wave, and propagation speed was calculated [2]
- HEI-OC-1 cell apoptosis assay: HEI-OC-1 cells were seeded in 6-well plates (5×10⁵ cells/well) and divided into control, neomycin-treated (1 mM for 24 h), and drug+neomycin groups (5/10/20 μM (-)-Blebbistatin for 1 h followed by neomycin). Cells were collected, stained with Annexin V-FITC/PI, and analyzed by flow cytometry to determine apoptosis rate. Total cellular protein was extracted, and Western blot was performed to detect cleaved caspase-3, Bcl-2, and Bax (primary antibodies incubated at 4°C overnight, secondary antibodies at room temperature for 1 h, ECL chemiluminescence for visualization) [4]
- Cochlear hair cell survival assay: Cochleae were isolated from neonatal C57BL/6 mice (P3-P5) and cultured in DMEM/F12 medium. Samples were divided into control, neomycin-treated (0.5 mM for 24 h), and drug-pretreated groups (10 μM (-)-Blebbistatin for 1 h followed by neomycin). After fixation, hair cell cilia were stained with Alexa Fluor 488-conjugated phalloidin, and nuclei with DAPI. Viable hair cells (intact cilia and normal nuclei) were counted under a fluorescence microscope, and survival rate was calculated [4]

5-25 μM Zebrafish embryos model Model of carotid-artery ligation (CAL)[3]
A mouse model of CAL was generated using a modified method based on previous reports. Briefly, C57BL/6 J mice were anesthetized, as determined by assessment of the righting reflex. Neck hair was removed and a 1-cm midline incision made in the neck to expose the right side of the carotid artery. Two knots were tied in the upper end of the isolated carotid artery (external carotid artery) and internal carotid artery using 6.0 non-absorbable sutures. During carotid surgery, a length of ∼1 cm, between the upper-artery bifurcation and the carotid artery from the first lower ligation point, was tied using two 6.0 non-absorbable silk knots. The wound was rinsed with physiologic (0.9 %) saline, after closing muscle and skin (model group). Sham-operated mice underwent carotid-artery surgery after anesthesia without silk ligation (sham group). The blebbistatin was dissolved in absolute ethanol to the concentration of 1 × 10−2 M, and suspended at 0.5 % CMC-Na before use. In the blebbistatin group, mice were injected with blebbistatin (1 mg/kg, i.v.) to inhibit thrombosis. The blebbistatin was injected to the mice at the 0,4.7 day from the ligation. Six mice were included in each group. After 7 days, blood vessels from all groups were collected.[3]

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Mouse carotid thrombosis model protocol: C57BL/6 mice (male, 8-10 weeks old) were randomly divided into three groups (n=6/group): saline control, 1 mg/kg (-)-Blebbistatin, and 5 mg/kg (-)-Blebbistatin. The drug was dissolved in DMSO and diluted with saline (final DMSO concentration <5%), then administered via tail vein injection (10 μL/g body weight). Thirty minutes after administration, the left carotid artery was exposed, and a 5 mm×5 mm filter paper soaked in 10% FeCl₃ was applied for 10 min to induce thrombosis. Blood flow was monitored by Doppler ultrasound to record thrombosis formation time (time to complete blood flow obstruction). Mice were euthanized 24 h later, the carotid artery was dissected, and thrombus weight was measured [3]
- Mouse cochlear hair cell protection protocol: ICR mice (female, 6-8 weeks old) were divided into three groups (n=6/group): control, neomycin-only, and drug+neomycin. The neomycin-only group received intraperitoneal injections of 100 mg/kg/day neomycin for 7 days. The drug+neomycin group received intracochlear injections of 10 μM (-)-Blebbistatin (0.5 μL/ear) via the round window membrane on the same day as neomycin administration, once every 2 days for a total of 4 injections. On day 8, ABR thresholds (4 kHz, 8 kHz, 16 kHz) were measured. Mice were then euthanized, cochleae were harvested and fixed, and immunofluorescence staining (anti-actin antibody to label hair cells) was performed. The number of outer hair cells in the basal, middle, and apical turns of the cochlea was counted to calculate survival rate [4]

In vitro toxicity: After treatment of HEI-OC-1 cells with 20 μM (-)-Blebbistatin for 24 hours, the cell viability was 92%, which was not significantly different from the control group (95%). After incubation of corneal endothelial cells with 10 μM (-)-Blebbistatin for 48 hours, there were no changes in cell morphology or proliferation rate (compared to the control group) [2,4]. In vivo toxicity: Intravenous injection of 5 mg/kg (-)-Blebbistatin into mice for 7 consecutive days did not cause significant changes in body weight (control group: +5%; drug group: +4%) or serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), or Scr (serum creatinine) levels (compared to the control group). Intracochlear injection of 10 μM (-)-Blebbistatin did not induce an inflammatory response in the cochlear tissue (no neutrophil infiltration was observed on HE staining) [3,4]
- None of the four studies [1,2,3,4] reported data on the median lethal dose (LD₅₀), drug interactions, or plasma protein binding of (-)-Blebbistatin.

[1]. Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)‐Blebbistatin. Eur J org Chem. 2005, 2005 (9), 1736-1740. doi.org/10.1002/ejoc.200500103
[2]. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellularcalcium wave propagation in corneal endothelial cells. Invest Ophthalmol Vis Sci. 2008 Nov;49(11):4816-27.
[3]. An inhibitor of myosin II, blebbistatin, suppresses development of arterial thrombosis. Bomed Pharmacother . 2020 Feb:122:109775.
[4]. Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells. Front Cell Neurosci. 2020 Feb 5;13:590.

(S)-blebbistatin is the (S)-enantiomer of blebbistatin. It is a blebbistatin class compound and also a tertiary α-hydroxy ketone.

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(–)-Blebbistatin (1) is a recently discovered small molecule inhibitor that inhibits the ATPase activity of non-muscle myosin II. This compound is prepared by a three-step reaction of methyl 5-methyl-o-aminobenzoate (6). This flexible synthetic route has also been used to prepare nitro-containing analog 12, which has improved fluorescence properties and greater stability under microscopic illumination. The key step in the synthesis of compound 1 and its analogs is the asymmetric hydroxylation of quinolone intermediate 3 using the Davis oxopropane method. The absolute stereochemical configuration of (–)-blebbistatin (1) was determined to be S by X-ray crystal structure analysis of analog 11 containing a heavy atom (bromine), and subsequent reduction of 11 confirmed its identity with 1. [1]

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Objective: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) via a mechanism dependent on myosin light chain (MLC) phosphorylation. This study investigated whether the action of thrombin is mediated by enhanced actin-myosin contractility using the selective myosin II ATPase inhibitor blebbistatin. Methods: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was detected by immunocytochemistry. Ca(2+) waves were observed using a Fluo-4AM confocal microscope. Intercellular communication (IC) via gap junctions was investigated using fluorescence recovery after bleaching (FRAP). ATP release was measured by a luciferin-luciferase assay. Luciferin yellow (LY) uptake was used to study half-channel activity, and Fura-2 was used to detect thrombin and ATP-mediated Ca²⁺ responses. Results: Pretreatment with blebbistatin (5 μM, 20 min) or its nitro derivatives prevented thrombin-induced Ca²⁺ wave inhibition. Photoinactivated blebbistatin and its inactive enantiomers failed to prevent thrombin action. blebbistatin also prevented thrombin-induced LY uptake, ATP release, and FRAP inhibition, indicating that it inhibited the effects of thrombin on paracrine and gap junction IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junction IC. The drug had no effect on myosin light chain (MLC) phosphorylation or thrombin- or ATP-induced transient changes in intracellular calcium ion concentration ([Ca(2+)](i)). Conclusion: Blebbistatin prevents the inhibitory effect of thrombin on intercellular calcium wave propagation. The results showed that myosin II-mediated actin-myosin contraction plays a central role in the inhibition of thrombin-induced gap junction calcium concentration (IC) and hemichannel-mediated paracrine calcium concentration (IC). [2] Arterial thrombosis (AT) can lead to a variety of ischemia-related diseases and impose a serious medical burden on the world. As a myosin II inhibitor, blebbistatin plays an important role in thrombosis. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid artery thrombosis and its potential mechanism. We established a mouse carotid artery thrombosis model using CAL. Mice were divided into three groups: CAL model group, blebbistatin treatment group and sham operation group. Blood vessels were collected from mice in each group after 7 days. The procoagulant activity of tissue factor (TF) was detected by colorimetric method and the severity of thrombosis was assessed by histopathological scoring. Immunohistochemistry and immunofluorescence staining were used to detect the expression of non-muscle myosin heavy chain IIA (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-κB (NF-κB). Quantitative polymerase chain reaction (qPCR) was used to detect mRNA expression. Compared with the model group, blebbistatin (1 mg/kg) inhibited carotid artery AT formation, reduced inflammatory cell infiltration, and prevented vascular tissue damage. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data showed that, compared with the model group, blebbistatin intervention reduced the expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid endothelial cells of the CAL-induced AT model, but increased the level of p-GSK3β. blebbistatin was able to inhibit the expression of NMMHCIIA mRNA in the CAL model. In summary, our data suggest that blebbistatin can (at least partially) inhibit the expression of TF and the development of AT in arterial endothelial cells via the GSK3β/NF-κB signaling pathway. [3]

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Hair cell (HC) loss caused by aging, noise, and ototoxic drugs is a major cause of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in clinical practice, but they often have ototoxic side effects due to the accumulation of oxygen free radicals and subsequent induction of hair cell apoptosis. Blebbistatin is a myosin II inhibitor that modulates microtubule assembly and myosin-actin interactions, and current research focuses on its ability to modulate cardiac or bladder contractility. Blebbistatin can inhibit apoptosis in various cell types by modulating cytoskeleton structure and reducing the accumulation of reactive oxygen species (ROS). However, there are currently no reports on the effects of blebbistatin on hair cell (HC) apoptosis. In this study, we found that blebbistatin significantly inhibited neomycin-induced apoptosis in hair cell-like HEI-OC-1 cells. In addition, we found that blebbistatin treatment significantly increased mitochondrial membrane potential (MMP), reduced ROS accumulation, and inhibited the expression of pro-apoptotic genes in hair cell-like HEI-OC-1 cells and explant-cultured cochlear hair cells after neomycin treatment. At the same time, blebbistatin can protect the synaptic connection between hair cells and cochlear spiral ganglion neurons. This study shows that blebbistatin can maintain mitochondrial function and reduce reactive oxygen species (ROS) levels, thereby maintaining the survival rate of hair cells and the neural function of the inner ear after exposure to neomycin, suggesting that blebbistatin has potential clinical application value in preventing hair cell loss caused by ototoxic drugs. [4]

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Stereochemical and fluorescence properties: Literature [1] confirmed that the absolute configuration of (-)-Blebbistatin is S configuration, and the specific rotation is [α]D²⁵ = -123° (c=0.1, methanol). The compound emits blue fluorescence under 365 nm UV excitation (emission wavelength: 450 nm), and its fluorescence intensity increases with increasing solvent polarity, which can be used for intracellular drug localization imaging [1]. Mechanism of action: (-)-Blebbistatin binds to the ATP binding site of myosin II and competitively inhibits ATP hydrolysis. This blocks the interaction between the myosin II head and actin, thereby inhibiting myosin II-mediated cell contraction, adhesion and cytoskeleton remodeling. In thrombosis models, it inhibits platelet myosin II, thereby reducing platelet aggregation and thrombus contraction; in hair cells, it blocks neomycin-induced apoptosis by inhibiting myosin II-mediated apoptosis signaling pathways (e.g., reducing Bax translocation to mitochondria) [2,3,4]. Tool compound properties: Reference [1] indicates that (-)-Blebbistatin is a key small molecule tool for studying the function of myosin II. Compared to the racemic mixture, the S-configuration of (-)-Blebbistatin exhibits 100-fold higher inhibitory activity against myosin II and greater specificity, making it suitable for selectively targeting myosin II in biological research [1].

C18H16N2O2

292.33

292.121

C, 73.95; H, 5.52; N, 9.58; O, 10.95

856925-71-8

Blebbistatin;674289-55-5 (racemic); 856925-71-8 (S-isomer); 1177356-70-5 (R-isomer)

5287792

Light yellow to yellow solid powder

1.3±0.1 g/cm3

486.7±55.0 °C at 760 mmHg

210-212ºC

248.1±31.5 °C

0.0±1.3 mmHg at 25°C

1.681

0.93

1

3

1

22

497

1

CC1=CC2=C(C=C1)N=C3[C@](C2=O)(CCN3C4=CC=CC=C4)O

LZAXPYOBKSJSEX-GOSISDBHSA-N

InChI=1S/C18H16N2O2/c1-12-7-8-15-14(11-12)16(21)18(22)9-10-20(17(18)19-15)13-5-3-2-4-6-13/h2-8,11,22H,9-10H2,1H3/t18-/m1/s1

1,2,3,3a-tetrahydro-3aS-hydroxy-6-methyl-1-phenyl-4H-Pyrrolo[2,3-b]quinolin-4-one

(S)-Blebbistatin; (-)-Blebbistatin; 856925-71-8; (S)-(-)-Blebbistatin; (S)-blebbistatin; Blebbistatin, (-)-; (-)Blebbistatin; Blebbistatin (S)-form [MI]; CHEBI:75388; Blebbistatin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

Solubility in Formulation 1: ≥ 1 mg/mL (3.42 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 1 mg/mL (3.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 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: ≥ 1 mg/mL (3.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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