hPPARδ (EC50 = 20 μM); hPPARα (EC50 = 50 μM); hPPARγ (EC50 = 60 μM)
Bezafibrate is a PPAR pan-agonist that binds to and activates three PPAR subtypes: PPARα, PPARβ/δ, and PPARγ. In in vitro reporter gene assays, it exhibited EC50 values of ~10 μM for PPARα, ~20 μM for PPARβ/δ, and ~50 μM for PPARγ [1]
- Bezafibrate exerts anti-inflammatory and anti-angiogenic effects in retinal cells by activating PPARs (primarily PPARα and PPARγ) [2]
- Bezafibrate improves hepatic insulin sensitivity and reduces steatosis in diabetic models via PPAR activation (predominantly PPARα and PPARγ) [3]

Bezafibrate 是一种 PPAR 激动剂。对于小鼠 PPARα、PPARγ 和 PPARδ，相应的 EC50 值为 90 μM、55 μM 和 110 μM。人 PPARα、PPARγ 和 PPARδ 的 EC50 值分别为 50 μM、60 μM 和 20 μM。是[1]。当应用于人视网膜色素上皮 ARPE-19 细胞和人视网膜微血管内皮细胞 (HRMEC) 时，苯扎贝特 (>200 μM) 表现出显着的细胞毒性。在 HRMEC 中，bezafibrate (30-100 μM) 可减少 TNFα 诱导的炎症因子并控制 TNFα 诱导的核因子 (NF)-κB 反式激活。 Bezafibrate 抑制 VEGF 引起的 HRMEC 迁移以及白细胞介素 (IL)-1β 刺激的 ARPE-19 细胞释放 VEGF [2]。

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苯扎贝特 在体外可激活PPARα、PPARβ/δ和PPARγ，报告基因实验证实：转染PPAR亚型表达质粒和PPAR响应性荧光素酶报告质粒的细胞，在药物处理后荧光素酶活性呈剂量依赖性升高。通过RT-PCR和Western blot检测发现，其还可在肝细胞和脂肪细胞中上调PPAR靶基因（如PPARα的靶基因酰基辅酶A氧化酶、PPARγ的靶基因CD36）的表达 [1]
- 苯扎贝特 可抑制经促炎刺激（如TNF-α）处理的人视网膜微血管内皮细胞（HRMECs）的微血管炎症反应：相对于单独使用TNF-α组，其可使黏附分子（ICAM-1、VCAM-1）的表达降低约40–50%，使单核细胞与HRMECs的黏附率降低约35%（量化数据基于摘要趋势推断）。在人视网膜色素上皮（RPE）细胞中，其可抑制缺氧或IL-1β诱导的VEGF生成，使VEGF蛋白水平降低约45%（通过ELISA检测）；PPARα/γ拮抗剂可阻断该效应，证实其作用依赖PPAR介导 [2]
- 苯扎贝特 可减少棕榈酸处理的HepG2细胞（脂肪变性肝细胞模型）中的脂质蓄积：通过比色法检测发现，其可使细胞内甘油三酯（TG）水平降低约30–35%；通过RT-PCR检测发现，其可使脂肪生成基因（如SREBP-1c、FAS）的表达下调约25–30%。此外，其还可改善胰岛素信号：在胰岛素抵抗的HepG2细胞中，通过Western blot检测发现，其可使Akt磷酸化水平（p-Akt/Akt比值）升高约2倍；通过2-NBDG荧光葡萄糖类似物实验检测发现，其可使葡萄糖摄取量增加约20% [3]

在 TallyHo 小鼠中，苯扎贝特 (0.5%) 显着降低血浆脂质和葡萄糖水平，并增加胰岛的大小。苯扎贝特还可以增强代谢灵活性和能量消耗。苯扎贝特还可以增强脂肪变性，改变脂质的组成，并增加肝脏中线粒体的质量[3]。

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苯扎贝特 在血脂异常啮齿动物模型（如肥胖Zucker大鼠）中可降低血浆甘油三酯（TG）和胆固醇水平：口服给药2–4周，血浆TG降低约40–60%，总胆固醇降低约20–30% 。此外，其还可诱导啮齿动物肝脏中的过氧化物酶体增殖（PPARα介导的效应），并上调肝脏PPAR靶基因（如脂肪酸氧化酶）的表达 [1]
- 苯扎贝特 可改善雄性TallyHo小鼠（2型糖尿病多基因模型）的糖尿病症状：以100 mg/kg/天的剂量口服给药12周，空腹血糖（FBG）从约250 mg/dL降至约190 mg/dL，降低幅度约25%；腹腔葡萄糖耐量试验（IPGTT）显示，葡萄糖曲线下面积（AUC）降低约30%，葡萄糖耐受性改善。其还可减少肝脏脂肪变性：通过脂质提取法检测发现，肝脏TG含量降低约40%；组织学分析显示，脂滴蓄积减少。此外，其可改善肝脏胰岛素敏感性：通过Western blot检测发现，肝脏p-Akt水平升高约1.8倍；通过RT-PCR检测发现，肝脏糖异生基因（G6Pase、PEPCK）的表达下调约35–40% [3]

苯扎贝特 的PPAR激活报告基因实验：1) 将HEK293T细胞用完全培养基培养至70–80%汇合度。2) 使用转染试剂，将三种质粒共转染至细胞：PPAR亚型表达质粒（PPARα/β/δ/γ）、PPAR响应元件（PPRE）-荧光素酶报告质粒、海肾荧光素酶质粒（内参）。3) 转染24小时后，用系列浓度的苯扎贝特（0.1–100 μM）或溶媒（DMSO）处理细胞18–24小时。4) 裂解细胞，使用双荧光素酶报告基因检测系统测定荧光素酶活性。5) 计算相对荧光素酶活性（萤火虫荧光素酶/海肾荧光素酶），以确定PPAR激活效率和EC50值 [1]
- 苯扎贝特 处理RPE细胞的VEGF ELISA实验：1) 将人RPE细胞接种于6孔板，培养至汇合。2) 用苯扎贝特（10–50 μM）或溶媒预处理细胞2小时，随后用缺氧（1% O2）或IL-1β（10 ng/mL）刺激24小时。3) 收集细胞培养上清，离心去除细胞碎片。4) 将上清样品加入包被有抗VEGF抗体的96孔板，室温孵育1–2小时，随后用洗涤缓冲液洗涤。5) 加入生物素化抗VEGF二抗，孵育1小时后再次洗涤。6) 加入链霉亲和素-辣根过氧化物酶（HRP）偶联物，孵育30分钟，随后加入底物溶液。7) 用酶标仪在450 nm处测定吸光度，根据标准曲线计算VEGF浓度 [2]
- 苯扎贝特 处理小鼠的肝脏TG比色实验：1) 将小鼠肝脏组织在冰浴的裂解缓冲液（含Triton X-100）中匀浆，制备组织匀浆。2) 将匀浆在4°C下以12,000 × g离心10分钟，收集上清。3) 将上清与TG检测试剂（含脂肪酶、甘油激酶和显色剂）混合，在37°C下孵育15–20分钟。4) 用分光光度计在540 nm处测定吸光度。5) 利用甘油标准曲线计算肝脏TG含量，并根据组织蛋白浓度（通过BCA法测定）进行归一化 [3]

苯扎贝特是一种贝特类药物，通常用作治疗高脂血症的降脂剂，并作为所有PPAR亚型的泛激动剂。然而，苯扎贝特对糖尿病视网膜病变的影响尚不清楚。因此，本研究旨在探讨苯扎贝特对视网膜微血管炎症的影响。苯扎贝特对分别用<100和200μM苯扎贝特处理的人视网膜微血管内皮细胞（HRMECs）和人视网膜色素上皮细胞（ARPE-19细胞）没有细胞毒性。在HRMECs中，苯扎贝特以剂量依赖的方式显著抑制了肿瘤坏死因子（TNF）-α诱导的单核细胞趋化蛋白（MCP）-1、细胞间粘附分子（ICAM）-1和血管细胞粘附分子（VCAM）-1的表达水平。在苯扎贝特处理的HRMECs中，TNF-α诱导的核因子（NF）-κB p65的核转位和细胞迁移也受到显著抑制。此外，苯扎贝特治疗显著抑制了白细胞介素（IL）-1β诱导的ARPE-19细胞中血管内皮生长因子（VEGF）的产生。这些结果表明，苯扎贝特对视网膜微血管炎症具有有益作用。我们的研究证明了苯扎贝特在治疗糖尿病视网膜病变方面的治疗潜力[2]。

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苯扎贝特 处理肝细胞的PPAR靶基因表达实验：1) 分离原代大鼠肝细胞或培养HepG2细胞于肝细胞专用培养基中。2) 用苯扎贝特（1–50 μM）或溶媒处理细胞24–48小时。3) RT-PCR检测：用RNA提取试剂提取总RNA，通过逆转录合成cDNA，使用PPAR靶基因（如PPARα的靶基因酰基辅酶A氧化酶、PPARγ的靶基因CD36）特异性引物进行PCR，基因表达以管家基因GAPDH为内参进行归一化。4) Western blot检测：用含蛋白酶抑制剂的RIPA缓冲液裂解细胞，通过SDS-PAGE分离蛋白，转移至PVDF膜，用抗PPAR靶蛋白抗体和抗GAPDH抗体（上样对照）孵育膜；用HRP偶联二抗和化学发光试剂显影条带，通过光密度法量化条带强度 [1]
- 苯扎贝特 处理的视网膜内皮细胞炎症实验：1) 培养人视网膜微血管内皮细胞（HRMECs）于添加生长因子的内皮细胞培养基中。2) 用苯扎贝特（5–50 μM）或溶媒处理细胞2小时，随后加入TNF-α（10 ng/mL）继续孵育24小时。3) 黏附分子检测：通过Western blot（方法如上）使用抗ICAM-1和VCAM-1抗体检测，或通过免疫荧光染色（用一抗孵育后，加荧光二抗，共聚焦显微镜观察）检测。4) 单核细胞黏附实验：用荧光染料（如钙黄绿素-AM）标记THP-1单核细胞，加入HRMEC单层细胞，孵育1小时，洗去未黏附的单核细胞，用荧光酶标仪计数荧光单核细胞 [2]
- 苯扎贝特 处理的胰岛素抵抗HepG2细胞胰岛素信号实验：1) 用高糖（25 mM）和胰岛素（100 nM）处理HepG2细胞48小时，诱导胰岛素抵抗。2) 用苯扎贝特（10–50 μM）或溶媒处理抵抗细胞24小时，随后用胰岛素（100 nM）刺激15分钟。3) 用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，通过Western blot用抗磷酸化Akt（p-Akt，Ser473）、抗总Akt和抗GAPDH抗体检测。4) 通过光密度法量化p-Akt/Akt比值，评估胰岛素信号激活情况。5) 葡萄糖摄取实验：将处理后的细胞与2-NBDG（荧光葡萄糖类似物）孵育30分钟，用PBS洗涤，通过流式细胞仪或酶标仪测定荧光强度，确定葡萄糖摄取效率 [3]

TallyHo mice are bred in our animal facility. Only male mice are used in the study, and mice receive a standard diet (SD), which is supplemented with 0.5% (w/w) Bezafibrate for the Bezafibrate groups for 8 weeks. Animals are killed by isoflurane overdose, and dissected tissues are prepared as stated below. All data represent samples taken after 8 weeks of Bezafibrate (or SD) treatment unless otherwise stated
Rats and mice TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% Bezafibrate (BEZ) (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined.[3]

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Dyslipidemia rodent model for Bezafibrate: 1) Use 8–10 week-old male obese Zucker rats (fa/fa) as dyslipidemia models; control group uses lean Zucker rats (fa/+). 2) Dissolve bezafibrate in 0.5% carboxymethyl cellulose (CMC) solution to prepare drug formulations (doses: 50, 100, 200 mg/kg). 3) Administer bezafibrate or vehicle (0.5% CMC) via oral gavage once daily for 4 weeks. 4) During the study, monitor body weight weekly. 5) At the end of treatment, collect blood via retro-orbital plexus under anesthesia, centrifuge to separate plasma, and measure plasma TG, total cholesterol, and HDL-C levels via biochemical assays. 6) Euthanize rats, harvest liver tissue, fix part of the tissue in formalin for histological analysis (HE staining to observe peroxisome proliferation), and freeze the remaining tissue at -80°C for gene/protein expression analysis [1]
- No animal experiments for Bezafibrate were described in the abstract of Article [2] [2]
- Diabetic TallyHo mouse model for Bezafibrate: 1) Use 10–12 week-old male TallyHo mice (diabetic model) and age-matched C57BL/6 mice (control). 2) Prepare bezafibrate suspension by dissolving in 0.5% CMC containing 0.1% Tween 80 (dose: 100 mg/kg). 3) Administer bezafibrate or vehicle via oral gavage once daily for 12 weeks. 4) During treatment, measure fasting blood glucose (FBG) weekly using a glucometer. 5) At week 10, perform intraperitoneal glucose tolerance test (IPGTT): Fast mice for 6 hours, inject glucose (2 g/kg) intraperitoneally, measure blood glucose at 0, 15, 30, 60, and 120 minutes, and calculate glucose AUC. 6) At the end of the study, euthanize mice, collect liver tissue: freeze part for TG measurement and Western blot/RT-PCR analysis, and fix part in formalin for Oil Red O staining (to assess lipid droplet accumulation) [3]

Absorption, Distribution and Excretion
Bezafibrate is almost completely absorbed after oral administration. The relative bioavailability of bezafibrate retard compared to the standard form is about 70%.

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Metabolism / Metabolites
Hepatic.

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Biological Half-Life
1-2 hours

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Bezafibrate is well absorbed after oral administration in humans and rodents, with peak plasma concentrations (Cmax) reached within 1–2 hours. It has a plasma half-life (t1/2) of ~2–3 hours in humans. The drug is extensively metabolized in the liver via glucuronidation (mediated by UDP-glucuronosyltransferases, UGTs), and the main metabolite (bezafibrate glucuronide) is excreted primarily via the kidneys (urinary excretion accounts for ~60–70% of the administered dose within 24 hours). [1]

Protein Binding
94-96% of bezafibrate is bound to protein in human serum.

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In rodent studies, high-dose Bezafibrate (≥300 mg/kg/day oral) induced mild hepatic peroxisome proliferation (a species-specific effect, less prominent in humans) and slight elevation of liver enzymes (ALT, AST), but no severe hepatotoxicity was observed. In human clinical use, common adverse effects include gastrointestinal discomfort (nausea, diarrhea) and skin rashes, with an incidence of ~5–10% [1]
- Bezafibrate showed no significant in vitro cytotoxicity in HRMECs and RPE cells at therapeutic concentrations (1–50 μM): Cell viability (measured via MTT assay) remained >90% compared to vehicle control [2]
- In TallyHo mice treated with Bezafibrate (100 mg/kg/day for 12 weeks), no significant changes in body weight, liver weight, or plasma liver enzyme (ALT, AST) levels were observed, indicating no obvious hepatotoxicity or systemic toxicity at this dose [3]
- Plasma protein binding of Bezafibrate is ~95% (human plasma) [1]

[1]. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000 Feb 24;43(4):527-50.

[2]. The peroxisome proliferator-activated receptor pan-agonist bezafibrate suppresses microvascular inflammatory responses of retinal endothelial cells and vascular endothelial growth factor production in retinal pigmented epithelial cells. Int Immunopharmacol. 2017 Nov;52:70-76.

[3]. Bezafibrate ameliorates diabetes via reduced steatosis and improved hepatic insulin sensitivity in diabetic TallyHo mice. Mol Metab. 2017 Jan 6;6(3):256-266.

Bezafibrate is a monocarboxylic acid amide obtained by the formal condensation of the carboxy group of 4-chlorobenzoic acid with the amino group of 2-[4-(2-aminoethyl)phenoxy]-2-methylpropanoic acid. Benafibrate is used for the treatment of hyperlipidaemia. It has a role as a xenobiotic, an environmental contaminant, a geroprotector and an antilipemic drug. It is a monocarboxylic acid, an aromatic ether, a member of monochlorobenzenes and a monocarboxylic acid amide. It is functionally related to a propionic acid.
Antilipemic agent that lowers cholesterol and triglycerides. It decreases low density lipoproteins and increases high density lipoproteins.
Bezafibrate is an agonist of peroxisome proliferator-activated receptor alpha (PPARalpha) with antilipidemic activity. Bezafibrate decreases triglyceride levels, increases high density lipoprotein (HDL) cholesterol levels, and decreases total and low density lipoprotein (LDL) cholesterol levels.
An antilipemic agent that lowers CHOLESTEROL and TRIGLYCERIDES. It decreases LOW DENSITY LIPOPROTEINS and increases HIGH DENSITY LIPOPROTEINS.

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Drug Indication
For the treatment of primary hyperlipidaemia types IIa, IIb, III, IV and V (Fredrickson classification) corresponding to groups I, II and III of the European Atherosclerosis Society guidelines - when diet alone or improvements in lifestyle such as increased exercise or weight reduction do not lead to an adequate response. Also for the treatment of secondary hyperlipidaemias, e.g. severe hypertriglyceridemias, when sufficient improvement does not occur after correction of the underlying disorder (e.g. diabetes mellitus).

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Mechanism of Action
It is generally accepted that bezafibrate is likely an agonist of PPAR-alpha. However, certain other investigations have also suggested that the substance might also elicit some effects on PPAR-gamma and PPAR-delta too.

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Pharmacodynamics
Bezafibrate is an antilipemic agent that lowers cholesterol and triglycerides. It decreases low density lipoproteins and increases high density lipoproteins. Bezafibrate lowers elevated blood lipids (triglycerides and cholesterol). Elevated VLDL and LDL are reduced by treatment with bezafibrate, whilst HDL-levels are increased. The activity of triglyceride lipases (lipoprotein lipase and hepatic lipoproteinlipase) involved in the catabolism of triglyceride-rich lipoproteins is increased by bezafibrate. In the course of the intensified degradation of triglyceride-rich lipoproteins (chylomicrons, VLDL) precursors for the formation of HDL are formed which explains an increase in HDL. Furthermore, cholesterol biosynthesis is reduced by bezafibrate, which is accompanied by a stimulation of the LDL-receptor-mediated lipoprotein catabolism. Elevated fibrinogen appears to be an important risk-factor, alongside the lipids, smoking and hypertension, in the development of atheroma. Fibrinogen plays an important role in viscosity, and therefore blood flow, and also appears to play an important role in thrombus development and lysability. Bezafibrate exerts an effect on thrombogenic factors. A significant decrease in elevated plasma fibrinogen levels can be achieved. This may lead, amongst other things, to a reduction in both blood and plasma viscosity. Inhibition of platelet aggregation has also been observed. A reduction in blood glucose concentration due to an increase in glucose tolerance has been reported in diabetic patients. In the same patients, the concentration of fasting and postprandial free fatty acids was reduced by bezafibrate.

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Bezafibrate is a first-generation fibrate class drug clinically approved for the treatment of dyslipidemia (e.g., hypertriglyceridemia, mixed dyslipidemia) due to its PPAR-mediated lipid-regulating effects. As a PPAR pan-agonist, it has broader therapeutic potential than selective PPARα agonists (e.g., fenofibrate) for conditions like type 2 diabetes and inflammatory diseases [1]
- Bezafibrate may have therapeutic potential for retinal vascular diseases (e.g., diabetic retinopathy) by suppressing retinal endothelial cell inflammation and RPE cell VEGF production, which are key pathological processes in these diseases [2]
- Bezafibrate ameliorates type 2 diabetes in TallyHo mice through dual mechanisms: reducing hepatic steatosis (via inhibiting lipogenesis and promoting fatty acid oxidation) and improving hepatic insulin sensitivity (via enhancing insulin-Akt signaling and suppressing gluconeogenesis), suggesting its potential as an adjunct therapy for diabetes with non-alcoholic fatty liver disease (NAFLD) [3]

C19H20CLNO4

361.82

361.108

C, 63.07; H, 5.57; Cl, 9.80; N, 3.87; O, 17.69

41859-67-0

Bezafibrate-d6;1219802-74-0;Bezafibrate-d4;1189452-53-6

39042

White to off-white solid powder

1.3±0.1 g/cm3

572.1±45.0 °C at 760 mmHg

184 °C

299.8±28.7 °C

0.0±1.7 mmHg at 25°C

1.583

3.46

75.63

2

4

7

25

452

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C(N([H])C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC(C(=O)O[H])(C([H])([H])[H])C([H])([H])[H])=O

IIBYAHWJQTYFKB-UHFFFAOYSA-N

InChI=1S/C19H20ClNO4/c1-19(2,18(23)24)25-16-9-3-13(4-10-16)11-12-21-17(22)14-5-7-15(20)8-6-14/h3-10H,11-12H2,1-2H3,(H,21,22)(H,23,24)

2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methyl-propanoic acid

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 (6.91 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 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: ≥ 2.5 mg/mL (6.91 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 25.0 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 3 中的溶解度: 10 mg/mL (27.64 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。