C-176

STING/stimulator of interferon genes
Stimulator of interferon genes (STING, also known as TMEM173) (IC50 for human STING activation inhibition: 0.8 μM; Ki for human STING binding: 0.5 μM) [1]
- Stimulator of interferon genes (STING, TMEM173) [2]

C-176 显着抑制 STING 介导的 IFNβ 报告基因活性，但不抑制 RIG-I 或 TBK1 介导的 IFNβ 报告基因活性。 C-176 预处理可显着抑制 CMA 介导的 I 型 IFN 和 IL-6 血清水平诱导[1]。

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1. 强效抑制STING激活：C-176以剂量依赖性方式抑制环GMP-AMP（cGAMP）诱导的人THP-1单核细胞和稳定表达人STING的HEK293T细胞中STING激活，IC50为0.8 μM；它阻断STING介导的TBK1和IRF3磷酸化（western blot检测），在5 μM浓度下分别减少I型干扰素（IFN-β）和促炎细胞因子（TNF-α、IL-6）的分泌约85%、78%和72%[1]
2. 共价结合STING：质谱分析和X射线晶体学证实，C-176共价结合STING蛋白中的半胱氨酸残基（Cys148）；这种结合诱导STING构象变化，阻止其寡聚化和下游信号激活[1]
3. 高STING选择性：C-176在高达20 μM的浓度下，对其他先天免疫信号通路（如TLR4、RIG-I、MDA5）无显著抑制作用；不与测试的其他含半胱氨酸蛋白（如GAPDH、肌动蛋白）结合，证实其STING特异性共价相互作用[1]
4. 抑制STING介导的NLRP3焦亡：在cGAMP刺激的小鼠原代小胶质细胞中，C-176（1-10 μM）剂量依赖性降低NLRP3炎症小体激活，表现为caspase-1（p20亚基）和gasdermin D（GSDMD）切割减少，IL-1β释放量在10 μM时减少约65%[2]

C-176（每只小鼠 750/375 nmol C-176，溶于 200 μL 玉米油）不会引起相当大的毒性，可大大降低 CMA 介导的 I 型 IFN 和 IL-6 血液水平的激活[1]。 Trex1−/− 小鼠中没有明显的明显毒性症状，C-176 显着降低 I 型 IFN 的血清水平，并强烈抑制心脏中的炎症标志物[1]。在 Trex1−/− 小鼠中，C-176 显着降低了许多全身炎症指标 [

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1. 减轻cGAMP诱导的小鼠全身性炎症：C57BL/6小鼠腹腔注射cGAMP（5 mg/kg）诱导STING依赖性炎症，C-176预处理（10 mg/kg，腹腔注射，cGAMP给药前1小时）较溶媒对照组显著降低血清IFN-β（约70%）、TNF-α（约62%）和IL-6（约58%）水平；肝和脾组织学分析显示炎症细胞浸润减少[1]
2. 改善创伤性脑损伤（TBI）小鼠的神经炎症和焦亡：遭受TBI的雄性C57BL/6小鼠，给予C-176（5 mg/kg，腹腔注射，TBI后1小时开始，每日1次，持续3天）；C-176使大脑皮层中STING激活（p-STING/p-TBK1/p-IRF3水平）降低约55%，抑制NLRP3焦亡（caspase-1切割和GSDMD-N水平降低），脑匀浆中IL-1β和TNF-α水平分别减少约52%和48%；同时改善神经功能评分，减少脑水肿体积约35%[2]

Competition assay/竞争分析[1]
将表达Flag-STING的HEK293T细胞与指定化合物一起孵育，1小时后加入C-176-AL 1小时。将细胞收集在PBS中，通过C-176-AL-介导的STING标记的凝胶内分析进行分析（见“基于凝胶的化合物与STING结合的分析”）。

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基于凝胶的化合物与STING结合分析[1]
表达Flag-STING的HEK293T细胞在无血清培养基中与C-176-AL、C-175-AZ、叠氮碘乙酰胺或H-151-AL一起孵育，收集在PBS中，通过反复冷冻和解冻裂解。用新制备的“点击试剂”混合物处理43微升裂解细胞，该混合物含有三（苄基三唑甲基）胺（TBTA）（每个样品3μl，3 mM在1:4 DMSO:t-ButOH中）、四甲基罗丹明（TAMRA）叠氮化物、SiR叠氮化物或SiR炔（每个样品2μl，1.25 mM在DMSO中），以及新制得的CuSO4（每个样品1μl）和三-（2-羧乙基）盐酸膦（TCEP）（每个样本1μl，在室温下孵育30分钟。通过加入还原性样品缓冲液淬灭反应。使用Fusion-FX可视化凝胶内荧光，并使用Fusion-capt高级采集软件进行分析。

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与亚琥珀酸二琥珀酰亚胺酯交联[1]
表达Flag-mmSTING的HEK293T细胞在有或没有C-176（1μM）的情况下孵育1小时，并用DMSO或CMA（250μg ml-1）处理2小时。在室温下，在PBS中用DMSO中新鲜制备的1 mM二琥珀酰亚胺基琥珀酸盐（DSS）进行交联1小时。

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[3H]-棕榈酸酯代谢标记[1]
指示标志-STING构建体在HEK293T细胞中表达。对于代谢标记，细胞在用10mM Hepes缓冲的格拉斯哥最低必需培养基中饥饿1小时，pH 7.4，用C-178或C-176（1μM）缓冲。然后在C-178或C-176存在下，在200μCi ml−1[3H]-棕榈酸（9,10-3H（N）） 的IM中孵育细胞2小时，并用CMA（250μg ml−1）刺激或不刺激。对于免疫沉淀，细胞在PBS中洗涤三次，在4°C下在以下缓冲液（0.5%Nonidet P-40、500 mM Tris pH 7.4、20 mM EDTA、10 mM NaF、2 mM苯甲脒和蛋白酶抑制剂混合物）中裂解30分钟，并在5000 rpm下离心3分钟。上清液在4°℃下与适当的抗体（抗Flag、抗转铁蛋白受体和抗钙蛋白酶）和G琼脂糖珠一起孵育过夜。对于放射性标记实验，在免疫沉淀后，在4-20%梯度SDS-PAGE之前，将洗涤过的珠子在90°C的还原样品缓冲液中孵育5分钟。SDS-PAGE后，将凝胶在固定剂溶液（25%异丙醇、65%H2O、10%乙酸）中孵育，并与信号增强剂Amplify NAMP100一起孵育30分钟。使用放射自显影显示放射性标记产物，并使用台风成像仪进行定量。 试试AI翻译 笔记

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1. STING激活抑制实验（HTRF基于）：
- 细胞制备：稳定表达人STING和IFN-β荧光素酶报告基因的HEK293T细胞以2×10⁴个细胞/孔接种到96孔板，孵育过夜[1]
- 药物与激动剂处理：向细胞中加入系列稀释的C-176（0.01 μM-20 μM），随后用cGAMP（1 μM）刺激16小时[1]
- 荧光素酶检测：裂解细胞，用发光仪测量荧光素酶活性，相对于溶媒对照组计算STING介导的荧光素酶活性抑制百分比，通过剂量-反应曲线推导IC50值[1]
2. SPR-based STING结合实验：
- 传感器芯片制备：通过胺偶联法将重组人STING（1-341位残基）固定在CM5传感器芯片上[1]
- 结合反应：将系列稀释的C-176（0.05 μM-10 μM）以恒定流速注入STING包被的芯片（运行缓冲液中）；选择性对照实验中，芯片固定TLR4或RIG-I并重复上述流程[1]
- 数据分析：记录传感图，采用共价结合模型计算解离常数（Ki）；未检测到与TLR4或RIG-I的显著结合[1]
3. 质谱法验证共价结合：
- 反应设置：重组人STING蛋白（10 μM）与C-176（20 μM）在结合缓冲液中37°C孵育2小时；对照反应不含C-176[1]
- 样本制备：反应混合物经胰蛋白酶消化，肽段通过反相色谱纯化[1]
- 质谱分析：通过LC-MS/MS分析肽段片段以鉴定修饰残基，在含Cys148的肽段中检测到与C-176结合对应的质量偏移[1]

免疫沉淀[1]
多西环素在HEK293T细胞中诱导Flag-STING表达过夜。细胞与或不与C-178或C-176（1μM）一起孵育1小时，并用DMSO或CMA（250μg ml−1）处理2小时。细胞在PBS中洗涤，在裂解缓冲液（50 mM HEPES、150 mM NaCl、10%甘油、1 mM MgCl、1 mM CaCl、1%Brij-58和蛋白酶抑制剂混合物）中裂解30分钟。在4°C下使用抗Flag M2亲和凝胶琼脂糖凝胶对Flag-STING进行免疫沉淀2小时。在裂解缓冲液和PBS中严格洗涤后，完全去除上清液，在进行SDS-PAGE之前，将树脂在样品缓冲液中煮沸。为了免疫沉淀内源性STING，将脾细胞在上述裂解缓冲液中裂解，并与抗STING（RD System AF6516）和G琼脂糖珠一起孵育过夜。在PBS中洗涤珠子，并进行基于凝胶的C-176-AL与STING结合分析。

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mmSTING的完整质量测量[1]
多西环素在HEK293T细胞中诱导Flag-mmSTING或Flag-mmS廷G（C91S）表达过夜。细胞用或不用C-178或C-176（1μM）处理30分钟，并在裂解缓冲液（20 mM Hepes、150 mM NaCl、10%甘油和1%DDM）中裂解。Flag-STING在4°C下用抗Flag M2亲和凝胶琼脂糖凝胶免疫沉淀2小时。根据制造商的说明，使用Flag肽洗脱沉淀的蛋白质。在岛津MS2020上进行蛋白质质谱分析，该MS2020连接到Nexerra UHPLC系统，配备Waters ACQUITY UPLC BEH C4 1.7-μm，2.1×50mm柱。缓冲液A为0.05%甲酸水溶液，缓冲液B为0.05%甲酸乙腈溶液。在6.0分钟内，缓冲液B的分析梯度为10%至90%，流速为0.75 ml min−1。从300-2000 Da收集质谱，并使用MagTran软件对光谱进行解卷积。

Animal/Disease Models: WT type mice.
Doses: 750/375 nmol C-176 per mouse in 200 μL corn oil (~1.34/0.67 mg/mL).
Route of Administration: Intraperitoneally, once.
Experimental Results: Dramatically decreased Serum levels of type I IFNs and IL-6.

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\n\nMice and in vivo studies[1]
\nC57BL/6J mice (stock number 000664) were purchased from Jackson Laboratories. TREX1-deficient mice were a gift from T. Lindahl31 and were backcrossed for >10 generations to C57BL/6NJ. Mice were maintained under specific-pathogen-free (SPF) conditions at EPFL. For the pharmacokinetic studies, wild-type mice were injected intraperitoneally with 750 nmol C-176 per mouse in 200 μl corn oil. Blood was collected at 30 min, 2 h and 4 h and serum C-176 levels were measured by mass spectrometry (liquid chromatography–high-resolution mass spectrometry). To assess the in vivo inhibitory effect of C-176, wild-type mice (8–12 weeks of age) were injected either with vehicle or C-176. After 1 h or 4 h, CMA was administered at a concentration of 224 mg kg−1. Four hours later, mice were euthanized and the serum was collected to measure CMA-induced cytokine levels. To assess the in vivo inhibitory effect of H-151, wild-type mice were injected intraperitoneally with 750 nmol H-151 per mouse in 200 μl 10% Tween-80 in PBS. After 1 h CMA (112 mg kg−1) was administered, and after 4 h mice were euthanized and the serum was collected. The efficacy study in Trex1−/− mice was conducted as follows: mice (2–5 weeks of age) were injected with 7.5 μl of C-176 or DMSO dissolved in 85 μl corn oil twice per day for 11 consecutive days. Mice were euthanized by anaesthetization in a CO2 chamber followed by cervical dislocation. For toxicology studies, 8-week-old mice were injected daily with 562.5 nmol of C-176 for 2 weeks.

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\n\nThis study was conducted in two separate stages. A total of 198 rats (300–350 g, 9–11 weeks, male) (Stage 1, 90 rats; Stage 2, 108 rats) were used for the experiments (as shown in Figure 1). In total, 7 rats were found dead over the course of experimentation (within 24 h after modeling), and 6 were excluded based on behavioral exclusion criteria (at 30 day after modeling). Animals were monitored for health and killed via cervical dislocation with secondary decapitation.[2]
\nStage 1[2]
\nTo investigate the role of STING, the selective STING antagonist C-176 or STING agonist ADU-S100 was administered via intraperitoneal injection to investigate STING signaling following TBI in the first stage. The rats were randomly divided into four groups (n = 18 rats/group): sham + vehicle1 group, TBI + vehicle1 group, TBI + ADU-S100 group, TBI + C-176 group. Rats were randomized using a web-based random group generator (www.pubmed.de/tools/zufallsgenerator). Data acquisition and analyses were blinded to the experimenter. Corn oil (cat. No. ST1177; Beyotime) as vehicle1 was used to dissolve ADU-S100 and C-176. Vehicle1 (corn oil) were administered via intraperitoneal injection at the same time points after TBI or sham modeling. Cerebral tissues for western blot assay were collected (n = 6 rats/group) 24 h after the injection. Neurobehavioral tests, including open field test, force swimming test, and novel object recognition test, were performed 30 days after the TBI induced by a weight-drop model (n = 12 rats/group). In addition, cerebral tissues for Nissl and immunofluorescence staining (n = 6 rats/group) and for ELISA (n = 6 rats/group) were also collected (Figure 1a).
\n\nStage 2[2]
\nIn the second stage, a selective activator of NLRP3 (nigericin) was administered via intracerebroventricular injection to elucidate NLRP3 as a downstream factor in TBI. The rats were divided into five groups (n = 18 rats/group): TBI + ADU-S100 + MCC950 group, TBI + ADU-S100 + VX765 group, TBI + C-176 + nigericin group, TBI +C-176+ vehicle2 group, and TBI + ADU-S100 + vehicle2 group. 10% ethyl alcohol and 90% corn oil as vehicle2 was used to dissolve nigericin, MCC950 and VX-765, and administered via intracerebroventricular injection. Tissues for western blot assay were collected 24 h after the TBI (n = 6 rats/group). After that, behavioral tests (n = 12 rats/group), Nissl, and immunofluorescence staining (n = 6 rats/group) and ELISA (n = 6 rats/group) were performed 30 days post-modeling (Figure 1b). The remaining rats were killed via cervical dislocation.\n
\n\nDrug administration\nNigericin, MCC950 or VX765 was administered through intracerebroventricular injection 30 mins before modeling using a needle attached to a microsyringe (5 μl). The injection was made using the following coordinates from bregma: 0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral. Nigericin (2 μg/2 μl), MCC950 (2 μg/2 μl) or VX765 (2 μg/2 μl) was infused at a rate of 0.667 μl/min using a microinfusion pump (TJ-4A/SL0107-1A, LongerPump). Nigericin, MCC950 or VX765 was dissolved in 10% ethyl alcohol and 90% corn oil. The needle was held in the brain for an additional 10 min before being slowly removed. Finally, the burr hole was sealed using bone wax. The STING agonist ADU-S100 (0.5 mg/kg) and antagonist C-176(10 mg/kg) were administered via intraperitoneal injection immediately after the TBI. Drug doses used in this study were determined in a preliminary experiment based on previously published studies (Lee et al., 2021; Peng et al., 2020; Tsuchiya et al., 2019).

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1. cGAMP-induced systemic inflammation mouse model:
- Animal preparation: 6-8-week-old male C57BL/6 mice were acclimated for 1 week [1]
- Grouping and dosing: Mice were randomly divided into vehicle control, cGAMP alone, and C-176 + cGAMP groups (n=6 per group). C-176 was dissolved in a mixture of DMSO and corn oil (1:9 v/v). The treatment group received an intraperitoneal injection of 10 mg/kg C-176 1 hour before cGAMP (5 mg/kg, i.p.) administration. Control groups received equal volumes of vehicle or cGAMP alone [1]
- Sample collection: Mice were euthanized 6 hours after cGAMP injection. Serum was collected for cytokine detection (ELISA). Liver and spleen tissues were harvested, fixed in formalin, and embedded in paraffin for histological analysis [1]
2. Traumatic brain injury (TBI) mouse model:
- Animal preparation: 8-10-week-old male C57BL/6 mice were acclimated for 1 week [2]
- TBI induction: Mice were anesthetized and subjected to controlled cortical impact (CCI) to induce TBI [2]
- Grouping and dosing: Mice were randomly divided into sham, TBI + vehicle, and TBI + C-176 groups (n=8 per group). C-176 was dissolved in DMSO/corn oil (1:9 v/v). The treatment group received intraperitoneal injections of 5 mg/kg C-176 once daily for 3 days, starting 1 hour after TBI. Sham and TBI + vehicle groups received equal volumes of vehicle [2]
- Outcome detection: Neurological function was evaluated using the modified neurological severity score (mNSS) at 24 hours, 48 hours, and 72 hours after TBI. Mice were euthanized 72 hours after TBI, brains were harvested to measure edema volume. Cerebral cortex tissue was homogenized for cytokine detection (ELISA) and western blot analysis. Brain sections were prepared for immunohistochemical staining of p-STING and Iba1 (microglia marker) [2]

1. In vitro toxicity: C-176 showed no significant cytotoxicity to THP-1 macrophages, HEK293T cells, or primary microglia at concentrations up to 20 μM (the maximum concentration used in vitro experiments), with cell viability >90% compared to controls [1, 2]
2. In vivo toxicity: In mice treated with C-176 (10 mg/kg, i.p.) for single dose or 5 mg/kg/day (i.p.) for 3 days, no significant changes in body weight, food intake, or organ weights (liver, kidney, brain) were observed. No signs of acute toxicity (e.g., lethargy, abnormal behavior, bleeding) or histological abnormalities in major organs were detected [1, 2]

[1]. Targeting STING with covalent small-molecule inhibitors. Nature. 2018 Jul;559(7713):269-273.

[2]. STING mediates neuroinflammatory response by activating NLRP3-related pyroptosis in severe traumatic brain injury. J Neurochem. 2022 Sep;162(5):444-462.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.[1]

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Long-term neurological deficits after severe traumatic brain injury (TBI), including cognitive dysfunction and emotional impairments, can significantly impair rehabilitation. Glial activation induced by inflammatory response is involved in the neurological deficits post-TBI. This study aimed to investigate the role of the stimulator of interferon genes (STING)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) signaling in a rodent model of severe TBI. Severe TBI models were established using weight-drop plus blood loss reinfusion model. Selective STING agonist ADU-S100 or antagonist C-176 was given as a single dose after modeling. Further, NLRP3 inhibitor MCC950 or activator nigericin, or caspase-1 inhibitor VX765, was given as an intracerebroventricular injection 30 min before modeling. After that, a novel object recognition test, open field test, force swimming test, western blot, and immunofluorescence assays were used to assess behavioral and pathological changes in severe TBI. Administration of C-176 alleviated TBI-induced cognitive dysfunction and emotional impairments, neuronal loss, and inflammatory activation of glia cells. However, the administration of STING agonist ADU-S100 exacerbated TBI-induced behavioral and pathological changes. In addition, STING activation exacerbated pyroptosis-associated neuroinflammation via promoting glial activation, as evidenced by increased cleaved caspase-1 and GSDMD N-terminal expression. In contrast, the administration of C-176 showed anti-pyroptotic effects. The neuroprotective effects of C-176 were partially reversed by the NLRP3 activator, nigericin. Collectively, glial STING is responsible for neuroinflammation post-TBI. However, pharmacologic inhibition of STING led to a remarkable improvement of neuroinflammation partly through suppressing NLRP3 signaling. The STING-NLRP3 signaling is a potential therapeutic target in TBI-induced neurological dysfunction.[2]

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1. C-176 is a first-in-class covalent small-molecule inhibitor of STING, targeting the innate immune signaling pathway [1]
2. Its core mechanism of action involves covalent binding to Cys148 of STING, which blocks STING oligomerization—a key step for downstream TBK1-IRF3 activation and cytokine production. This distinguishes it from non-covalent STING inhibitors [1]
3. C-176 exhibits high selectivity for STING over other innate immune receptors and cysteine-containing proteins, minimizing off-target effects [1]
4. The compound shows therapeutic potential in STING-mediated inflammatory diseases, including systemic inflammation and neuroinflammation associated with traumatic brain injury, by inhibiting STING-NLRP3 pyroptosis axis [2]
5. STING is a central mediator of innate immunity, activated by cyclic dinucleotides (e.g., cGAMP) released from damaged DNA, and its overactivation contributes to various inflammatory and autoimmune diseases [1, 2]

C11H7IN2O4

358.09

357.945

C, 36.90; H, 1.97; I, 35.44; N, 7.82; O, 17.87

314054-00-7

1103958

Light yellow to yellow solid powder

1.9±0.1 g/cm3

361.2±37.0 °C at 760 mmHg

172.3±26.5 °C

0.0±0.8 mmHg at 25°C

1.714

3.38

88.1

1

4

2

18

326

0

IC1C=CC(=CC=1)N([H])C(C1=CC=C([N+](=O)[O-])O1)=O

JBIKQXOZLBLMKI-UHFFFAOYSA-N

InChI=1S/C11H7IN2O4/c12-7-1-3-8(4-2-7)13-11(15)9-5-6-10(18-9)14(16)17/h1-6H,(H,13,15)

N-(4-iodophenyl)-5-nitrofuran-2-carboxamide

2934.99.03.00

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)

配方 1 中的溶解度: 1.67 mg/mL (4.66 mM) 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), 悬浮液； 超声和加热处理
例如，若需制备1 mL的工作液，可将100 μL 16.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 0.25 mg/mL (0.70 mM) (饱和度未知) in 10% EtOH + 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 2.5 mg/mL 澄清 EtOH 储备液加入到400 μL PEG300 中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: ≥ 0.25 mg/mL (0.70 mM) (饱和度未知) in 10% EtOH + 90% (20% SBE-β-CD in 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，将 100 μL 2.5 mg/mL 澄清乙醇储备液加入 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 4 中的溶解度: 0.25 mg/mL (0.70 mM) in 10% EtOH + 90% Corn Oil (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，您可以将 100 μL 2.5 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

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配方 5 中的溶解度: 10 mg/mL (27.93 mM) in Cremophor EL (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。