CA3 (CIL-56)

YAP/TEAD interaction
The target of CA3 (CIL-56) is Yes-associated protein 1 (YAP1)/Tead transcriptional complex; [1]

在体外，CA3显着减少食管腺癌细胞的生长。诱导细胞凋亡、减少肿瘤球形成和减少 ALDH1+ 细胞的数量都是 CA3 可能的作用。在 293T 细胞中共转染每个转录因子的单独启动子荧光素酶后，CA3 特异性抑制 Tead/YAP1 转录活性，但对 Super-TOP/Wnt、CBF1/Notch 或 AP-1 没有影响。抗辐射食管腺癌细胞中富含的 CSC 特性优先被 CA3 抑制[1]。

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1. 抑制YAP1/Tead转录活性：在共转染Gal4-Tead、5×UAS-荧光素酶和YAP1 cDNA的293T细胞中，与其他化合物（A414、A432、A413、A433、VP）相比，CA3 (CIL-56)处理可显著降低YAP1/Tead荧光素酶报告基因活性。此外，在SKGT-4和JHESO食管腺癌（esophageal adenocarcinoma, EAC）细胞中，通过相同的荧光素酶报告基因实验发现，CA3 (CIL-56)可呈剂量依赖性降低YAP1/Tead转录活性[1]
2. 对EAC细胞的抗增殖活性：4种EAC细胞系（SKGT-4、Flo-1、JHESO、OACP）经CA3 (CIL-56)（0.1µM、0.5µM、1µM）处理后，MTS实验结果显示，与0.1% DMSO对照组相比，细胞增殖和活力呈剂量依赖性抑制。对于经多西环素诱导YAP1过表达的SKGT-4细胞（DOX+），CA3 (CIL-56)的抗增殖效应比未诱导YAP1的细胞（DOX−）更显著[1]
3. 诱导细胞周期阻滞和凋亡：SKGT-4和JHESO细胞经CA3 (CIL-56)（0.5µM、1µM）处理24h和48h后，经碘化丙啶染色并通过流式细胞术分析。结果显示，与对照组相比，细胞周期时相分布发生改变（细胞周期阻滞），且肿瘤细胞死亡指数显著升高[1]
4. 下调YAP1和SOX9表达：免疫印迹分析表明，CA3 (CIL-56)可呈剂量依赖性降低SKGT-4和JHESO细胞中YAP1及其下游靶蛋白SOX9的表达水平。实时定量聚合酶链反应（quantitative real-time PCR, qPCR）进一步证实，CA3 (CIL-56)可降低这些细胞中YAP1的mRNA水平。免疫荧光染色也显示，经CA3 (CIL-56)处理的JHESO细胞中YAP1和SOX9的表达降低[1]
5. 抑制辐射抵抗性EAC细胞的癌症干细胞（cancer stem cell, CSC）特性：Flo-1 XTR（辐射抵抗性EAC细胞）比亲本Flo-1-P细胞具有更强的CSC特性（肿瘤球形成能力增强、ALDH1+细胞比例升高）。CA3 (CIL-56)呈剂量依赖性抑制Flo-1-P和Flo-1 XTR细胞的肿瘤球形成，且对Flo-1 XTR细胞的抑制效应更显著。此外，CA3 (CIL-56)（0.5µM处理48h）可降低Flo-1 XTR细胞中ALDH1+细胞的比例，并下调这些辐射抵抗性细胞中YAP1、磷酸化EGFR（phospho-EGFR）和磷酸化S6（phospho-S6）的蛋白水平[1]
6. 与5-氟尿嘧啶（5-FU）的协同作用：4种EAC细胞系（SKGT-4、JHESO、OACP、Yes-6）及YAP1过表达的SKGT-4（DOX+）细胞经CA3 (CIL-56)与5-FU单独或联合处理后，MTS实验显示，与单独用药或对照组相比，联合处理可显著增强细胞生长抑制作用，且在YAP1高表达的EAC细胞中协同效应更强[1]
7. 对YAP1/Tead通路的特异性：在转染YAP1/Tead、Super-TOP/Wnt、CBF1/Notch或AP-1通路报告质粒的293T细胞中，CA3 (CIL-56)（0.5µM、1µM）仅特异性抑制YAP1/Tead的荧光素酶活性，而不影响Wnt、Notch或AP-1通路的活性[1]

在异种移植模型中，CA3 表现出有效的抗肿瘤活性，且没有明显的毒性[1]。

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CA3在可诱导的高YAP异种移植小鼠体内模型中具有较强的抗肿瘤作用[1]
为了进一步证实CA3靶向YAP1在体内的抗肿瘤作用，我们利用可诱导的YAP1高SKGT-4 （Dox+）细胞异种移植模型。以每只小鼠1 × 106个细胞的剂量给裸鼠植入Dox -和Dox+ SKGT-4细胞。在这个浓度下，我们观察到在注射10-14天后，只有Dox+ SKGT-4细胞能够形成肿瘤（图5B），这表明YAP1是体内驱动肿瘤生长所必需的。为了确定CA3对yap诱导的肿瘤生长的抑制作用，我们将携带Dox+ SKGT-4 EAC异种移植物的小鼠随机分为两组，然后分别给予对照磷酸盐缓冲盐水或1mg /kg的CA3治疗。在我们3周的给药计划结束时，测量SKGT-4异种移植肿瘤的重量和体积以及小鼠的体重。体内SKGT-4 Dox+异种移植物模型的结果表明，CA3处理的Dox+ SKGT-4异种移植物小鼠体内肿瘤大小和重量大大减少（图5b&5c），而在整个实验期间，植入Dox - SKGT-4细胞的小鼠未形成肿瘤（图5B）。CA3处理组与对照组小鼠体重无显著差异（图5D）。此外，免疫组织化学在小鼠肿瘤组织中进一步证实，CA3处理sktt -4 Dox+后，YAP1、SOX9和KI67的表达显著降低（图5E）。因此，CA3在体内有效抑制EAC肿瘤生长，这些作用至少部分归因于抑制干性基因YAP1和SOX9。

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CA3与5-FU协同抑制EAC细胞体外和体内生长[1]
为了确定CA3单独或与5-FU联合处理对EAC细胞系生长的抑制作用，我们首先在96孔板中播种4个YAP1组成高表达的EC细胞系（sktt -4、JHESO、OACP和YES-6），并分别用CA3单独、5-FU单独或CA3与5-FU在指定浓度下联合处理。图6A的结果表明，尽管CA3对这四种细胞系的生长产生了剂量依赖性的降低，但CA3与5-FU的结合对其生长的抑制作用显著，尤其是CA3与5-FU的结合作用最大。为了进一步研究CA3对EAC细胞的抑制是否依赖于YAP1，我们用CA3单独、5-FU单独或联合处理Dox+ （YAP1诱导）和Dox - SKGT4 （PIN20YAP1）细胞，我们发现CA3单独与YAP1低SKGT-4细胞（Dox−）相比，优先抑制YAP1高SKGT-4细胞（Dox+）的生长，并且呈剂量依赖性（图6B）。此外，CA3和5-FU联合处理对Dox+和Dox−SKGT-4细胞的生长均产生最大的抑制作用（图6b）。这些结果表明，CA3与5-FU在GAC细胞生长抑制中的协同作用。

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1. 在YAP1诱导型异种移植模型中的抗肿瘤活性：将具有YAP1诱导表达能力的SKGT-4细胞（PIN20YAP1）皮下接种于裸鼠（每组5只，双侧接种）。小鼠分为有无多西环素（DOX）处理组（以诱导YAP1表达），并给予CA3 (CIL-56)或溶媒对照处理。结果显示，在DOX+组（YAP1过表达肿瘤）中，CA3 (CIL-56)处理显著降低肿瘤重量，且对小鼠体重无显著影响，表明其耐受性良好[1]
2. 在JHESO异种移植模型中的抗肿瘤活性：将JHESO细胞（1.5×10⁶个/位点）皮下接种于裸鼠（每组5只，双侧接种）。小鼠分别接受CA3 (CIL-56)单独处理、5-FU单独处理或两者联合处理。结果显示，联合处理组的肿瘤体积缩小程度显著大于单独用药组或溶媒对照组。肿瘤组织的免疫组织化学（immunohistochemistry, IHC）分析表明，CA3 (CIL-56)（单独或与5-FU联合）可降低YAP1、SOX9和Ki67（增殖标志物）的表达[1]

SOX9 荧光素酶报告基因之前已有描述。之前也描述过的 5 × -UAS-荧光素酶报告基因和 Gal4-TEAD4 构建体是从 MD 安德森癌症中心的 Johnson 博士获得的。如前所述，用 SOX9 荧光素酶报告基因和 Renilla 载体或 5 × -UAS-荧光素酶报告基因和 Gal4-TEAD4 与 CMV-β-gal 构建体瞬时共转染食管腺癌细胞。

将 SKGT-4 和 JHESO 细胞接种到 DMEM 中的 6 孔板（1 × 105/孔）中，培养 24 小时以允许细胞贴壁。然后将细胞按照推荐的不同剂量暴露于 0.1% DMSO（对照）或 CA3 中 48 小时。然后收集细胞，用甲醇固定，洗涤，用RNase A处理，用碘化丙啶进行DNA染色，并进行DNA直方图和细胞周期阶段分布的流式细胞术分析。

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细胞增殖试验[1]
用0.1%二甲亚砜（对照）、不同剂量的<强>CA3处理EAC细胞及其耐药对应细胞。对于联合处理实验，按指示用<强>CA3、5-FU或不同浓度的联合处理细胞6天，并使用前面描述的MTS法评估细胞活力。所有试验一式三份，至少重复三次。

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流式细胞术及凋亡分析[1]
采用流式细胞术对EAC细胞凋亡进行分析。简而言之，将SKGT-4和JHESO细胞在Dulbecco改良Eagle培养基中接种到六孔板上（每孔1 × 105），培养24小时以使细胞附着。然后用0.1%二甲亚砜（对照）或CA3按不同剂量处理细胞48小时。接下来，收集细胞，用甲醇固定，清洗，用RNase A处理，用碘化丙啶染色，用流式细胞仪分析细胞的DNA直方图和细胞周期分布。

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蛋白的提取和Western blot分析[1]
从经CA3处理的EAC细胞中分离出蛋白质，并按照前面描述的方法进行Western blotting分析。

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1. YAP1/Tead转录活性实验（荧光素酶报告基因实验）：将293T细胞或EAC细胞（SKGT-4、JHESO）共转染Gal4-Tead质粒、5×UAS-荧光素酶报告质粒和YAP1 cDNA质粒（以过表达YAP1）。转染后，用不同浓度（0.5µM、1µM或其他指定剂量）的CA3 (CIL-56)处理细胞48h，随后检测荧光素酶活性，以评估CA3 (CIL-56)对YAP1/Tead转录活性的抑制作用。为检测通路特异性，将细胞转染Super-TOP/Wnt、CBF1/Notch或AP-1通路的报告质粒，再经CA3 (CIL-56)处理并检测荧光素酶活性[1]
2. 细胞增殖/活力实验（MTS实验）：将EAC细胞（SKGT-4、Flo-1、JHESO、OACP、Yes-6，包括SKGT-4 DOX+和DOX−细胞、Flo-1-P和Flo-1 XTR细胞）接种于适宜的培养板中。细胞贴壁后，用CA3 (CIL-56)（0.1µM、0.5µM、1µM）单独、5-FU单独或两者联合（指定浓度）处理6天（协同实验）或其他指定时长。向各孔中加入CellTiter Aqueous One Solution（MTS试剂），检测490nm处的吸光度（OD490），并以0.1% DMSO对照组为参照，计算细胞增殖/活力百分比[1]
3. 细胞周期与凋亡分析（流式细胞术）：将SKGT-4和JHESO细胞接种于6孔板中，用CA3 (CIL-56)（0.5µM、1µM）或0.1% DMSO处理24h和48h。收集细胞、固定后用碘化丙啶染色，通过流式细胞术分析DNA直方图以确定细胞周期时相分布，并计算肿瘤细胞死亡指数[1]
4. 免疫印迹实验：用指定剂量的CA3 (CIL-56)处理EAC细胞（SKGT-4、JHESO、Flo-1-P、Flo-1 XTR），制备细胞裂解液，经凝胶电泳分离蛋白后转移至膜上，用抗YAP1、SOX9、phospho-EGFR、phospho-S6或其他相关蛋白的抗体进行孵育。采用标准免疫印迹流程进行检测，并对蛋白水平进行半定量分析[1]
5. 实时定量PCR（qPCR）：用指定剂量的CA3 (CIL-56)处理SKGT-4和JHESO细胞，提取总RNA并合成互补DNA（cDNA）。使用YAP1特异性引物进行qPCR，以合适的内参基因（如GAPDH）为参照，采用2⁻ΔΔCt法计算YAP1 mRNA的相对表达量[1]
6. 免疫荧光染色：用CA3 (CIL-56)或对照处理JHESO细胞，固定并透化后，加入抗YAP1和SOX9的一抗孵育，再用荧光标记的二抗孵育。通过共聚焦显微镜观察并定量YAP1和SOX9的表达[1]
7. 肿瘤球形成实验：将Flo-1-P和Flo-1 XTR细胞以低密度接种于超低吸附培养板中，在培养开始时加入指定浓度的CA3 (CIL-56)。培养8~10天后，在显微镜下计数直径>50µm的肿瘤球，拍摄球体制备图像，通过球体数量量化评估CSC的自我更新能力[1]
8. ALDH1+细胞比例分析：用ALDH1标记试剂盒对Flo-1、Flo-1 XTR细胞及经CA3 (CIL-56)（0.5µM处理48h）处理的Flo-1 XTR细胞进行染色，通过流式细胞术检测并定量ALDH1+细胞（CSC标志物）的百分比[1]

PBS; 1 mg/kg; i.p.
JHESO xenograft mice model of esophageal adenocarcinoma In vivo xenograft mouse model[1]
In vivo experiments have been conducted in accordance with an Institutional Animal Care and Use Committee (IACUC). SKGT-4 (PIN20YAP1) cells (1 × 106) without (Dox−) or with (Dox+) YAP1 induction by Doxycycline were inoculated into nude mice (n = 5/group). The mice in the Dox+ group were fed drinking water containing 2.5% sucrose and 2.5% Doxycycline, whereas those in the Dox− group were fed water containing only 2.5% sucrose. After 10 days, CA3 was introperitoneally injected into the animals in the Dox+ group at 1 mg/kg/mouse three times a week for total 3 weeks.
In a JHESO xenograft model of EAC, 2 × 106 JHESO cells were subcutaneously injected into nude mice (n = 5/group). After about 10 days, the mice underwent intraperitoneal injection of CA3 at 1 mg/kg/mouse, 5-FU at 30 mg/kg/mouse, or a combination of them three times a week for total 3 weeks. A control group was given phosphate-buffered saline at 100 µl/mouse. The mice’s tumor volumes, tumor weights, and body weights were measured as described previously (4). All measurements were compared using an unpaired Student t-test.

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1. YAP1-inducible SKGT-4 xenograft experiment:
- Cell preparation: SKGT-4 cells with inducible YAP1 expression (PIN20YAP1) were cultured and harvested in log-phase growth.
- Xenograft establishment: Nude mice (5 per group) were inoculated subcutaneously with SKGT-4 (PIN20YAP1) cells at two sites (left and right flanks) per mouse.
- Treatment groups: Mice were divided into groups with or without doxycycline (DOX) administration (to induce YAP1 expression) and treated with CA3 (CIL-56) or vehicle control. The specific dose of CA3 (CIL-56) and administration frequency/route were not explicitly stated, but treatment continued for 6 weeks.
- Endpoints: After 6 weeks, mice were euthanized, tumors were excised and weighed. Mouse body weight was measured regularly to monitor toxicity. Tumor tissues were collected for immunohistochemistry (IHC) analysis of YAP1, SOX9, and Ki67 [1]
2. JHESO xenograft experiment:
- Cell preparation: JHESO EAC cells were cultured, and 1.5×10⁶ cells per site were prepared for injection.
- Xenograft establishment: Nude mice (5 per group) were inoculated subcutaneously with JHESO cells at two sites (left and right flanks) per mouse.
- Treatment groups: Mice were randomly assigned to four groups: vehicle control, CA3 (CIL-56) alone, 5-FU alone, or CA3 (CIL-56) + 5-FU combination. The specific doses of CA3 (CIL-56) and 5-FU, as well as administration frequency/route, were not explicitly stated, but treatment was continued until the study endpoint.
- Endpoints: Tumor volume was measured regularly using calipers (tumor volume = length × width² / 2). Mouse body weight was monitored to assess tolerability. At the end of the study, tumor tissues were collected for IHC analysis of YAP1, SOX9, and Ki67 [1]

The only toxicity-related observation was that treatment with CA3 (CIL-56) (alone or in combination with 5-FU) did not cause a significant reduction in mouse body weight in both xenograft models, suggesting acceptable in vivo tolerability at the tested doses. No data on median lethal dose (LD50), hepatotoxicity, nephrotoxicity, drug-drug interactions, or plasma protein binding degree of CA3 (CIL-56) was provided in the literature [1]

[1]. Mol Cancer Ther . 2018 Feb;17(2):443-454.

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high-expressing esophageal adenocarcinoma cells both in vitro and in vivo Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443-54. ©2017 AACR.

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1. Background: Yes-associated protein 1 (YAP1), a coactivator in the Hippo pathway, is a key mediator of CSC properties, tumor progression, and therapy resistance in various cancers, including esophageal adenocarcinoma (EAC). YAP1 is often a terminal node of multiple oncogenic pathways, making it a potential therapeutic target. However, effective YAP1 inhibitors were previously lacking [1]
2. Mechanism of action: CA3 (CIL-56) exerts its antitumor effects primarily by specifically inhibiting the YAP1/Tead transcriptional complex. This inhibition leads to downstream effects, including downregulation of YAP1 and its target gene SOX9, suppression of CSC properties (reduced tumor sphere formation, decreased ALDH1+ cells), induction of cell cycle arrest and apoptosis, and inhibition of cell proliferation. In radiation-resistant EAC cells, CA3 (CIL-56) also downregulates phospho-EGFR and phospho-S6, which are associated with therapy resistance [1]
3. Therapeutic potential: CA3 (CIL-56) shows particular efficacy in YAP1-high EAC cells and radiation-resistant EAC cells (which have enhanced CSC properties). Its synergistic effect with 5-FU (a commonly used chemotherapeutic agent for EAC) suggests that CA3 (CIL-56) could be a promising candidate for the treatment of YAP1-driven, therapy-resistant EAC [1]
4. Chemical structure: The chemical structure of CA3 (CIL-56) is provided in Figure 1E of the literature [1]

C23H27N3O5S2

489.61

489.139

C, 56.42; H, 5.56; N, 8.58; O, 16.34; S, 13.10

300802-28-2

654092

White to off-white solid powder

1.5±0.1 g/cm3

741.7±70.0 °C at 760 mmHg

402.4±35.7 °C

0.0±2.6 mmHg at 25°C

1.714

3.66

124

1

8

4

33

870

0

S(C1C=CC2=C(/C(/C3C=C(C=CC2=3)S(N2CCCCC2)(=O)=O)=N/O)C=1)(N1CCCCC1)(=O)=O

XYZXEEIUKQGUHB-UHFFFAOYSA-N

InChI=1S/C23H27N3O5S2/c27-24-23-21-15-17(32(28,29)25-11-3-1-4-12-25)7-9-19(21)20-10-8-18(16-22(20)23)33(30,31)26-13-5-2-6-14-26/h7-10,15-16,27H,1-6,11-14H2

N-[2,7-bis(piperidin-1-ylsulfonyl)fluoren-9-ylidene]hydroxylamine

2934.99.9001

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: 2.5 mg/mL (5.11 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.08 mg/mL (4.25 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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: ≥ 2.08 mg/mL (4.25 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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 5% DMSO+40% PEG 300+5% Tween 80+50% ddH2O: 0.5mg/ml

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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。