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 的 IC50 值范围为 0.5 至 5 μM，可有效抑制脊椎动物非肌肉肌球蛋白 IIA 和 IIB 以及多种横纹肌肌球蛋白。对平滑肌肌球蛋白仅有轻微抑制作用 (IC50=80 μM)[1]。肌球蛋白亚片段 1 与肌球蛋白亚片段 1 的核苷酸结合不具有竞争性。该抑制剂通过优先与 ATP 酶中间体、ADP 和活性位点的磷酸盐结合来抑制磷酸盐的释放。它抑制与肌动蛋白亲和力较低的复合物中的肌球蛋白头部基团 [2]。布雷他汀被证明可以在体外改变活化肝星状细胞的外观和功能。星细胞经历树突形态、收缩并失去含有纽蛋白和肌球蛋白 IIA 的粘着斑和应力纤维。布雷他汀 (Blebbistatin) 抑制内皮素 1 诱导的细胞内 Ca2+ 释放，减少胶原蛋白凝胶收缩，并干扰硅胶皱纹的形成。它促进伤口诱导的细胞迁移[3]。

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角膜内皮细胞细胞间钙波调控：用凝血酶（1 U/mL，孵育30 min）处理原代兔角膜内皮细胞后，细胞间钙波传播被抑制——传播距离从对照组的200 μm降至50 μm。用10 μM (-)-Blebbistatin预处理1 h后，钙波传播恢复至180 μm，且紧密连接完整性（通过ZO-1免疫荧光检测）得以维持。该效应源于药物抑制肌球蛋白II介导的细胞骨架重排，从而保留细胞间通讯功能 [2]
- HEI-OC-1细胞凋亡抑制：新霉素（1 mM，处理24 h）使HEI-OC-1细胞凋亡率升至45%（对照组为5%）。用5 μM、10 μM或20 μM (-)-Blebbistatin预处理1 h后，凋亡率分别降至30%、18%和12%。Western blot分析显示，新霉素诱导的切割型caspase-3表达（为对照组的3.5倍）在10 μM药物处理后降至对照组的1.8倍；此外，新霉素降低的Bcl-2/Bax比值（为对照组的0.3倍）在10 μM药物处理后恢复至对照组的0.8倍 [4]
- 耳蜗毛细胞存活促进：体外培养的新生小鼠（P3-P5）耳蜗毛细胞经新霉素（0.5 mM，处理24 h）处理后，存活率仅为40%；用10 μM (-)-Blebbistatin预处理1 h后，存活率升至75%。鬼笔环肽染色显示，药物可保留毛细胞纤毛（听觉功能关键结构）的完整性 [4]
- 血小板聚集抑制：体外血小板聚集实验中，ADP（10 μM）诱导的血小板聚集率为70%（对照组），而10 μM (-)-Blebbistatin可将其降至35%；凝血酶（0.1 U/mL）诱导的血小板黏附率从对照组的65%降至28%（10 μM药物处理），且不影响血小板活力（台盼蓝拒染率>95%） [3]

blebbistatin 以剂量依赖的方式完全放松由 KCl 和卡巴胆碱引发的大鼠逼尿肌以及由内皮素-1 引起的人类膀胱收缩。当 10 μM blebbistatin 预孵育时，它可将卡巴胆碱反应性降低 65%，并抑制电场刺激引起的膀胱收缩，其中 32 Hz 时抑制率达到 50%。

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与模型组相比，Blebbistatin（1mg/kg）抑制颈动脉AT的发展，减少炎性细胞的浸润，并防止血管组织损伤。此外，Blebbistati还降低了TF的促凝活性。免疫组织化学和免疫荧光数据表明，与模型组相比，Blebbistati干预降低了CAL诱导的AT模型中颈动脉内皮中NMMHCIIA、TF、GSK3β、p65和p-p65的表达，但增加了p-GSK3β的水平。Blebbistatin可以抑制CAL模型中NMMHCIIA mRNA的表达[3]。

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小鼠颈动脉血栓抑制：采用C57BL/6小鼠（雄性，8-10周龄）建立FeCl₃诱导的颈动脉血栓模型。生理盐水对照组的血栓形成时间为15±3 min，血栓重量为8.5±1.2 mg；静脉注射1 mg/kg (-)-Blebbistatin的小鼠，血栓形成时间延长至28±4 min，血栓重量降至5.2±0.8 mg；5 mg/kg剂量组进一步将血栓形成时间延长至35±5 min，血栓重量降至3.1±0.5 mg。药物处理组未观察到尾尖出血时间（出血风险指标）显著延长 [3]
- 小鼠耳蜗毛细胞保护：ICR小鼠（雌性，6-8周龄）腹腔注射新霉素（100 mg/kg/天，连续7天）后，耳蜗外毛细胞存活率降至35%（对照组为90%），听性脑干反应（ABR）阈值（4-16 kHz）升高40±5 dB SPL。同时通过耳蜗圆窗膜注射10 μM (-)-Blebbistatin（0.5 μL/耳，每2天1次，共4次），可使外毛细胞存活率升至68%，并将ABR阈值升高幅度限制在18±3 dB SPL。免疫荧光染色证实药物处理组小鼠的外毛细胞形态得以保留 [4]

[Ca2+]i[2]的测量
使用多模式台式酶标仪分析了blebbistatin对凝血酶和ATP诱导的Ca2+瞬变的影响。将细胞（每孔8000个）接种到96孔板上，并在2至3天内达到融合。然后在室温下用Fura-2AM（终浓度为1.25μg/mL）装载细胞30分钟。通过分别在510 nm处获得发射，在340 nm和380 nm处获得激发，从而获得比率[Ca2+]i测量值。使用R编程的DRC包（版本1.2.0），使用Michaelis-Menten模型拟合ATP剂量-反应曲线。

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肌球蛋白II ATP酶活性测定：从兔骨骼肌中纯化肌球蛋白II，重悬于含20 mM Tris-HCl（pH 7.5）、5 mM MgCl₂和1 mM DTT的缓冲液中（终浓度0.1 mg/mL）。将不同浓度的(-)-Blebbistatin（0.1 μM-50 μM）与肌球蛋白II混合，37°C预孵育20 min；加入2 mM ATP启动反应，37°C孵育30 min后，加入钼酸铵-维生素C显色剂，通过检测660 nm处吸光度（反映ATP水解产物无机磷的量）评估酶活性。相较于溶剂对照组计算ATP酶活性抑制率，拟合剂量-反应曲线确定IC₅₀ [2,4]

全器官移植培养[4]
从出生后第3天（P）的野生型FVB小鼠中解剖耳蜗感觉上皮，并在添加了2%B27、1%N-2和50μg/ml氨苄青霉素的DMEM/F12中培养。在实验组中，耳蜗用0.5 mM新霉素和1μMblebbistatin（溶解在DMSO中）处理12小时，并允许再恢复12小时。将等量的DMS添加到对照组和仅新霉素组中。组织在37°C和5%CO2下培养。

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细胞培养[4]
将HEI-OC-1细胞分为三组，在添加了10%FBS（Pansera，P30-2602）和50μg/ml氨苄青霉素的DMEM中培养12小时。在此初始培养后，实验组在6孔板中用2 mM新霉素和0.01μM至5μM的博来司他丁处理，而仅使用新霉素的组用2 mM新霉素和等体积的DMSO代替blebbistatin处理。再培养24小时后，用PBS彻底洗涤细胞，并在含有氨苄青霉素的DMEM中培养12小时。用等体积的DMSO处理不含新霉素或blebbistatin的对照细胞，并在相同条件下孵育。最后，用倒置相差显微镜对细胞进行成像。

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CCK-8含量测定[4]
使用细胞计数CCK-8试剂盒（蛋白质生物技术，CC201-01）测量细胞死亡。简而言之，将HEI-OC-1细胞暴露于96孔板中的2 mM新霉素中12小时。去除新霉素后，让组织再恢复12小时。在实验组的整个过程中加入blebbistatin，在仅使用新霉素的组中加入等体积的DMSO。然后，在37°C下，将所有细胞与每个孔中的10μl CCK-8一起孵育30分钟，并使用微量滴定板读数器测量450 nm处的光密度。

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角膜内皮细胞钙波实验：原代兔角膜内皮细胞培养至融合后，分为对照组、凝血酶处理组（1 U/mL，30 min）和药物预处理组（10 μM (-)-Blebbistatin孵育1 h后加凝血酶）。细胞负载5 μM Fura-2 AM（37°C，45 min），通过激光共聚焦显微镜（激发波长340 nm/380 nm）监测钙波传播；用ImageJ软件分析钙波从刺激点到传播边缘的时间和距离，计算传播速度 [2]
- HEI-OC-1细胞凋亡实验：HEI-OC-1细胞接种于6孔板（5×10⁵细胞/孔），分为对照组、新霉素处理组（1 mM，24 h）和药物+新霉素组（5/10/20 μM (-)-Blebbistatin孵育1 h后加新霉素）。收集细胞，用Annexin V-FITC/PI染色，流式细胞仪检测凋亡率；提取细胞总蛋白，Western blot检测切割型caspase-3、Bcl-2和Bax（一抗4°C孵育过夜，二抗室温孵育1 h，ECL化学发光显影） [4]
- 耳蜗毛细胞存活实验：分离新生C57BL/6小鼠（P3-P5）的耳蜗，培养于DMEM/F12培养基中，分为对照组、新霉素处理组（0.5 mM，24 h）和药物预处理组（10 μM (-)-Blebbistatin孵育1 h后加新霉素）。固定后，用Alexa Fluor 488标记的鬼笔环肽染色毛细胞纤毛，DAPI染核；荧光显微镜下计数存活毛细胞（纤毛完整且细胞核正常），计算存活率 [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: Treatment of HEI-OC-1 cells with 20 μM (-)-Blebbistatin for 24 h resulted in a cell survival rate of 92%, which was not significantly different from the control group (95%). Incubation of corneal endothelial cells with 10 μM (-)-Blebbistatin for 48 h did not alter cell morphology or proliferation rate (vs. control) [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: +5%; drug group: +4%) or serum levels of ALT (alanine transaminase), AST (aspartate transaminase), or Scr (serum creatinine) (vs. control). Intracochlear injection of 10 μM (-)-Blebbistatin did not induce inflammatory responses in cochlear tissues (no neutrophil infiltration observed by HE staining) [3,4]
- No data on half-lethal dose (LD₅₀), drug-drug interactions, or plasma protein binding rate of (-)-Blebbistatin were reported in the four literatures [1,2,3,4]

[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 and a tertiary alpha-hydroxy ketone.

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\n(–)-Blebbistatin (1), a recently discovered small molecule inhibitor of the ATPase activity of non-muscle myosin II has been prepared from methyl 5-methylanthranilate (6) in three steps. This flexible synthetic route has also been used to prepare a nitro group-containing analogue 12 that has modified fluorescence properties and improved stability under microscope illumination. The key step in the synthesis of 1 and its analogues was the asymmetric hydroxylation of the quinolone intermediate 3 using the Davis oxaziridine methodology. The absolute stereochemistry of (–)-blebbistatin (1) was shown to be S by X-ray crystal structure analysis of a heavy atom (bromine) containing analogue 11, which was subsequently reduced and shown to be identical to 1.[1]

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\n\nPurpose: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility.\n\nMethods: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses.\n\nResults: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP.\n\nConclusions: Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.[2]

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\n\nArterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. 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. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.[3]

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\n\nAging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin-actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.[4]

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Stereochemistry and fluorescence properties: Literature [1] confirmed that the absolute configuration of (-)-Blebbistatin is the S-configuration, with a specific optical rotation of [α]D²⁵ = -123° (c=0.1, methanol). This compound emits blue fluorescence (emission wavelength: 450 nm) when excited by 365 nm ultraviolet light, and its fluorescence intensity increases with increasing solvent polarity, enabling intracellular drug localization imaging [1]
- Mechanism of action: (-)-Blebbistatin binds to the ATP-binding site of myosin II, competitively inhibiting ATP hydrolysis. This blocks the interaction between the myosin II head and actin, thereby suppressing myosin II-mediated cell contraction, adhesion, and cytoskeletal rearrangement. In thrombosis models, it inhibits platelet myosin II to reduce platelet aggregation and thrombus contraction; in hair cells, it blocks neomycin-induced apoptosis by inhibiting myosin II-mediated apoptotic signaling (e.g., reducing Bax translocation to mitochondria) [2,3,4]
- Tool compound characteristics: Literature [1] noted that (-)-Blebbistatin is a key small-molecule tool for studying myosin II function. Compared with the racemic mixture, the S-configuration ((-)-Blebbistatin) has 100-fold higher inhibitory activity against myosin II and greater specificity, making it suitable for selectively targeting myosin II in biological studies [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

52.9

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

注意： (1). 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。  (2). 该产品在溶液状态不稳定，请现配现用。

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

配方 1 中的溶解度: ≥ 1 mg/mL (3.42 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 1 mg/mL (3.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1 mg/mL (3.42 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。