AR 231453

GPR119

AR231453对所有其他测试的 GPCR（超过 230 个，包括所有已知的胰岛受体）均无活性，表明它对 GPR119 具有高度选择性[1]。 AR231453有效刺激 cAMP 积累 (EC50 = 4.7 nM)，其最大功效与毛喉素相似。 AR 231453 显着增强 HIT-T15 细胞中的胰岛素释放，EC50 为 3.5 nM[1]。 AR 231453 还在葡萄糖浓度范围为 8-17 mM 时刺激离体小鼠胰岛中的胰岛素释放[1]。

---

GPR119激动剂刺激胰岛素体外释放[1]
观察到GPR119与g - αs偶联并在β细胞中表达，提示我们验证GPR119是一种促胰岛素受体的假设。在这方面，AR231453显著增强了HIT-T15细胞中的胰岛素释放，EC50为3.5 nM（图4A），与其对cAMP的作用相似(EC50为4.7 nM；见图3D)，效果与福斯克林相似。为了评估GPR119对AR231453的胰岛素依赖性，我们测试了其在缺乏GPR119的RIN-5F细胞（RIN-5F/vector）和稳定转染了人GPR119的RIN-5F细胞（RIN-5F/hGPR119）中的活性。AR231453在RIN-5F/hGPR119细胞中刺激胰岛素释放，但在RIN-5F/载体细胞中没有（图4B）。这些研究表明，AR231453在表达gpr119的β细胞系中特异性地具有胰岛素促胰岛素作用。接下来，我们研究了GPR119激动剂对离体大鼠和小鼠胰岛中葡萄糖刺激的胰岛素释放的影响。在15 mM葡萄糖下，300 nM AR231453与GLP-1相似，增强了大鼠胰岛中的胰岛素释放（图4C）。然而，该化合物对5毫米葡萄糖培养的胰岛没有影响。因此，GPR119激动剂在胰岛中的胰岛素促胰岛素作用是葡萄糖依赖的。AR231453在葡萄糖浓度范围为8-17 mM时也刺激了离体小鼠胰岛的胰岛素释放（图4D）。这表明GPR119激动剂的胰岛素调节作用只需要适度的血糖升高。

AR231453（20 mg/kg，口服）以剂量依赖性方式显着改善口服葡萄糖耐量，其功效与磺酰脲格列本脲的最大有效剂量相似[1]。动物模型：小鼠[1]。剂量：20毫克/公斤。给药方法：口服，一次。结果：小鼠的葡萄糖耐量得到改善。[1]
GPR119激动剂改善小鼠葡萄糖耐量，并促进葡萄糖依赖性胰岛素释放[1]
AR231453在C57BL/6小鼠中表现出良好的口服生物利用度，在本研究使用的剂量下达到微摩尔血浆水平超过2小时。AR231453（口服20mg /kg）以剂量依赖的方式显著改善口服葡萄糖耐量，其疗效与磺脲格列本脲的最大有效剂量相似（图5A）。平均而言，AR231453最大限度地抑制血糖漂移约42%（21个独立实验）。当给糖时，AR231453改善葡萄糖耐量的效果稍差(23%；三个独立实验)（图5B）。

---

AR231453在gpr119缺陷小鼠中无活性[1]
为了证明AR231453的体内效应是通过GPR119发生的，我们制造了GPR119缺陷小鼠（图7）。与人类一样，小鼠GPR119基因位于X染色体上（Unigene Cluster Mm.34953）（图7A）。通过Southern blot分析（图7B）、胰腺GPR119 mRNA的RT-PCR分析（图7C）和敲除小鼠胰腺切片上GPR119受体的免疫荧光分析（图7D）证实了GPR119的缺失。gpr119缺陷小鼠的胰岛保持正常形态（图7D），对葡萄糖和GLP-1的反应正常（图7E）。这些小鼠也具有正常的大小、体重和进食/空腹血糖水平（未显示），并且在葡萄糖耐量试验中对格列本脲有典型的反应（图7，F和G）。因此，GPR119的缺失不会严重影响葡萄糖稳态。然而，来自这些动物的分离胰岛不再对AR231453有反应（图7E）。AR231453对GPR119缺陷小鼠的糖耐量也没有影响（图7G），表明观察到的AR231453在体内的活性确实是通过GPR119介导的。

---

方法：每只化学诱导的C57BL/6小鼠左肾下移植100只同种C57BL/6小鼠胰岛。从移植当天开始，这些受体每天给予溴脱氧尿嘧啶（BrdU），伴或不伴AR231453，剂量为10 mg/kg/d。通过测量血糖水平监测胰岛移植功能。4周时，行左肾切除术，切除携带胰岛移植物的肾脏，以测定胰岛移植物中β细胞的复制情况。胰岛素和BrdU免疫荧光染色检测复制的β细胞。共聚焦显微镜下对移植胰岛细胞中胰岛素（+）和BrdU(+) β细胞进行计数。为了确定AR231453是否会增加血浆GLP-1水平，我们在治疗后30分钟收集AR231453治疗小鼠的血浆，并通过酶联免疫吸附法测量血浆中GLP-1的活性。
结果：尽管所有受体小鼠在接受或不接受治疗的28天内达到血糖正常，但AR231453治疗的小鼠在更短的时间内达到血糖正常。小鼠在16±6天内达到血糖正常，而ar231453只需要8±3天（P < 0.01）。ar231453处理的小鼠胰岛移植物中胰岛素（+）和BrdU(+) β细胞的百分比明显高于载药处理的小鼠。ar231453处理小鼠胰岛移植物中胰岛素（+）和BrdU(+) β细胞的平均百分比为21.5%±6.9%，载药处理小鼠为5.6%±3.7% （P < 0.01）。ar231453处理小鼠血浆活性GLP-1水平也显著高于载药处理小鼠（P < 0.05）。
结论：我们的数据表明，GPR119激动剂AR231453可刺激β细胞复制，改善胰岛移植功能[2]。

体外测定[1]
cAMP测定采用Flash Plate腺苷酸环化酶试剂盒。简单地说，用Lipofectamine 转染HEK293细胞，用空载体DNA或GPR119表达质粒DNA（如上所述）。24 h后，在GIBCO细胞分离缓冲液（目录13151-014）中收获转染的细胞，并在实验缓冲液（50% 1× PBS/50%刺激缓冲液）中重悬。化合物在室温下每孔105个细胞孵育60分钟。在检测缓冲液中用示踪剂孵育2小时后，在Wallac MicroBeta闪烁计数器中计数。每个孔的cAMP值根据每个测定板上的标准cAMP曲线推断。在仓鼠胰岛素瘤来源的HIT-T15细胞和gpr119转染的RIN-5F稳定系中进行cAMP测定，基本上以相同的方式进行，但没有瞬时转染。在旨在选择性降低内源性GPR119在HIT-T15细胞中的表达的实验中，细胞被转染了一种对照RNA寡核苷酸，其序列来源于仓鼠GPR119的义取向（5 ' -CUAUGCUGCUAUCAAUCUA-3 ‘）（对照）或反义取向（5 ’ -UAGAUUGAUAGCAGCAUAG-3 '）[小干扰RNA (siRNA)]。转染48小时后，检测细胞GPR119表达和激动剂刺激的cAMP产生，如上所述。

为测定肌醇磷酸积累量，用指定的G蛋白受体表达质粒转染96孔板中的HEK293细胞。此外，细胞用巨细胞病毒（CMV）空载体或Gαq/Gαi表达质粒共转染，其中Gαq的末端6个残基被Gαi的相应残基取代。受体和G蛋白嵌合体分别以4:1的摩尔比转染。第二天，细胞在含0.4 μCi [3H]肌肌醇的100 μl无肌醇/无血清DMEM中孵育。细胞孵育过夜，然后用100 μl无肌醇/无血清DMEM（含10 μM pargyline和10 mM氯化锂）替换培养基。1小时后，细胞裂解，AG1-X8甲酸树脂层析分离磷酸肌醇。经过结合、四次洗涤和多屏过滤板洗脱等额外纯化后，用Wallac闪烁计数器定量洗脱计数。

为了体外胰岛素释放，HIT-T15胰岛素瘤细胞被镀在24孔板中（每孔2.5 × 105个细胞）进行胰岛素释放测定。实验前一天，将培养基改为DMEM （3 mM葡萄糖），其中含有10%的马血清和2.5%的胎牛血清。第二天，用PBS洗涤细胞两次，在0.25 ml hepes缓冲的Krebs-Ringer缓冲液中，在15mm葡萄糖存在下，与DMEM中的测试化合物孵育1小时。采用超灵敏胰岛素酶联免疫吸附测定试剂盒检测上清胰岛素水平。用RIN-5F稳定系进行胰岛素测定的方法基本相同。

使用雌性Sprague Dawley大鼠（体重，175-185 g）或雄性C57BL/6小鼠（体重，25 g）分离的胰岛进行胰岛素释放试验。如前所述，在静态胰岛孵育中测定胰岛素释放。简单地说，每组5个胰岛放入孵育井中。用含有5 mM葡萄糖的hepes缓冲克雷布斯-林格缓冲液（pH 7.4）预孵育30分钟后，将胰岛转移到含有2 ml hepes缓冲克雷布斯-林格缓冲液和不同浓度葡萄糖和测试化合物的井中。研究在37℃的水浴摇床中进行，气氛为95% O2/5% CO2。孵育缓冲液于60 min后收集样品，用ELISA法测定胰岛素。

In vivo experiments [1]
C57BL/6 male mice were used. For the oral glucose tolerance test, overnight fasted mice (n = 6 per treatment) were given either vehicle (80% polyethylene glycol 400/10% Tween 80/10% ethanol) or test compounds at desired doses via oral gavage. A glucose bolus was then delivered (3 g/kg orally or 2 g/kg ip). Plasma glucose levels were determined at desired time points over a 2-h period using blood (∼5 μl) collected from tail nick and a glucose meter. For insulin pharmacodynamic studies, vehicle or AR231453 was administered orally to fasted animals (n = 6 per treatment group and time point). After 30 min, a glucose bolus of 3 g/kg was administered orally. Blood was collected in heparinized blood collection tubes at desired time points. Plasma samples were obtained via centrifugation at 500 × g for 20 min and assayed for insulin as described above.

---

Islet Transplantation and Treatment [2]
The islets were isolated and transplanted according to a protocol that is similar to a previously published isolation protocol.6 Islets free of acinar cells, vessels, lymph nodes, and ducts were used for culture and transplantation. A total of 100 islets was transplanted into each recipient mouse. The recipient mice were randomly divided into two groups and treatments started from the day of transplantation. Mice in group 1 were orally treated with vehicle daily. Mice in group 2 were orally treated with AR231453 at 10 mg/kg/d. All recipient mice were also intraperitoneally treated with bromodeoxyuridine (BrdU) at 100 mg/kg/d for 4 weeks. After 4 weeks, nephrectomy was performed and all of the left kidneys bearing primary islet grafts were collected.

---

Plasma GLP-1 Active Immunoassay [2]
Mice were orally treated with vehicle or AR231453 at 10 mg/kg. At 30 minutes after treatment, blood samples were collected. Active GLP-1 in the plasma was measured using a mouse GLP-1 enzyme-linked immunosorbent assay kit.

[1]. A role for beta-cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucose-dependent insulin release. Endocrinology. 2007 Jun;148(6):2601-9.

[2]. Stimulating β-cell replication and improving islet graft function by AR231453, A GPR119 agonist. Transplant Proc. 2011 Nov;43(9):3217-20.

Pancreatic beta-cell dysfunction is a hallmark event in the pathogenesis of type 2 diabetes. Injectable peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor have shown significant promise as antidiabetic agents by virtue of their ability to amplify glucose-dependent insulin release and preserve pancreatic beta-cell mass. These effects are mediated via stimulation of cAMP through beta-cell GLP-1 receptors. We report that the Galpha(s)-coupled receptor GPR119 is largely restricted to insulin-producing beta-cells of pancreatic islets. Additionally, we show here that GPR119 functions as a glucose-dependent insulinotropic receptor. Unlike receptors for GLP-1 and other peptides that mediate enhanced glucose-dependent insulin release, GPR119 was suitable for the development of potent, orally active, small-molecule agonists. The GPR119-specific agonist AR231453 significantly increased cAMP accumulation and insulin release in both HIT-T15 cells and rodent islets. In both cases, loss of GPR119 rendered AR231453 inactive. AR231453 also enhanced glucose-dependent insulin release in vivo and improved oral glucose tolerance in wild-type mice but not in GPR119-deficient mice. Diabetic KK/A(y) mice were also highly responsive to AR231453. Orally active GPR119 agonists may offer significant promise as novel antihyperglycemic agents acting in a glucose-dependent fashion. [1]

---

Objective: G protein-coupled receptor 119 (GPR119) is predominantly expressed in β cells and intestinal L cells. AR231453 is a selective small-molecular GPR119 agonist that enhances glucose-dependent insulin secretion and glucagon-like peptide 1 (GLP-1) release. We investigated whether AR231453 can directly stimulate β-cell replication and improve islet graft function in diabetic mice.[2]

---

These data provide strong evidence that GPR119 is a significant modulator of glucose-stimulated insulin release in pancreatic β-cells. The GPR119-selective agonist AR231453 stimulated cAMP accumulation and insulin release in 1) transfected RIN-5F insulinoma cells, 2) HIT-T15 insulinoma cells that express the receptor endogenously, and 3) isolated rat and mouse islets. These effects were substantially the same as those elicited by forskolin or GLP-1, suggesting significant physiological relevance. In vivo, AR231453 stimulated insulin release and improved glucose tolerance with comparable efficacy to glyburide, a very robust stimulator of insulin release in rodents and humans. Importantly, the activity of AR231453 was particularly impressive in diabetic KK/Ay mice. All these effects are very likely occurring via GPR119. RIN-5F control cells lacking GPR119 are unresponsive to AR231453. In HIT-T15 cells, siRNA-mediated reduction in GPR119 levels was associated with virtually complete loss of responsiveness to AR231453. Finally, AR231453 does not enhance glucose-stimulated insulin release in islets derived from GPR119-deficient mice, nor does it improve glucose tolerance in GPR119-deficient mice. GPR119-deficient mice had no obvious defect in homeostasis, but this perhaps is not surprising because mice lacking either GIP receptor or GLP-1 receptor have very minor alterations in this regard. Because we did not measure AR231453 exposures in GPR119-deficient mice, its inactivity in glucose tolerance tests performed in these mice could simply be due to strain-dependent differences in plasma exposure of the compound. However, this seems unlikely. In C57BL/6 mice, AR231453 achieves exposures more than 100-fold greater than its in vitro EC50. Additionally, the compound is effective in all other strains of mice used here. Numerous, structurally distinct compounds have been assessed in wild-type littermates and GPR119-deficient mice, with similar outcomes to those seen for AR231453 (not shown). Taken together, these data strongly suggest that the actions of AR231453 are mediated via GPR119 and firmly establish that GPR119 is a physiologically significant mediator of glucose homeostasis. Although these studies support the hypothesis that GPR119 regulates glucose homeostasis by direct enhancement of pancreatic β-cell function, it is important to recognize that GPR119 may work via additional mechanisms. The effectiveness of AR231453 is reduced by almost 50% when glucose is administered ip, suggesting that its actions may also involve modulation of incretin signaling. This hypothesis warrants further investigation. Our studies in islets and mice indicate that GPR119 stimulates insulin release in a glucose-dependent manner. Even when administering doses of AR231453 (100 mg/kg in C57BL/6 mice) that greatly exceed the maximal in vivo efficacy of this compound, there is no observed stimulation of insulin release or lowering of glucose under fasted conditions. By contrast, glyburide elicited significant insulin release and marked hypoglycemia in fasted mice. These data are entirely consistent with the well-established mechanism by which GIP and GLP-1 stimulate insulin release.[1]

---

In this study, we found that AR231453 treatment significantly enhanced the reversal of diabetes in mice that received a marginally therapeutic dose of islets. It confirmed our earlier studies of using oleoylethanolamide (OEA), an endogenous GPR119 ligand, and PSN632408, a selective small-molecular GPR119 agonist, to enhance islet graft function.8 Reversal of diabetes depended on the function of the islet grafts since hyperglycemia reoccurred after removal of the left kidney bearing the islet grafts. We also found that AR231453 treatment significantly increased β-cell replication in islet grafts. In our previous study, we found that exendin-4 can stimulate β-cell replication in mouse islet grafts from both young and old donors.4 Therefore, we used islets from old donors for transplantation. Although it has been shown that the capacity for β-cell replication in aged mice is declined,our data showed that AR231453 could stimulate β-cell replication in islets isolated from old donors. AR231453 directly stimulates insulin secretion from β-cells in vitro and improves glucose tolerance and enhances glucose-dependent insulin release in vivo.1 Recently we also found that OEA and PSN632408 can directly stimulate β-cell replication in cultured islets (manuscript submitted for publication). Thus, AR231453 may improve islet graft function and stimulate β-cell replication in islet grafts by direct activation of GPR119 on islets. However, AR231453 can also stimulate GLP-1 secretion from intestinal enteroendocine L-cells through activation of GPR119. We found that AR231453 treatment increased the plasma GLP-1 levels in mice. Therefore, it is possible that AR231453-stimulated β-cell replication in islet grafts is also due to increased plasma GLP-1 concentrations. To address the direct and/or indirect effect of AR231453 through activating GRP119 on improving islet graft function and stimulating β-cell replication in islet grafts, GLP-1 receptor knockout mice and GPR119 knockout mice will have to be used as islet donors and/or transplant recipients. Targeting GPR119 is a novel therapeutic approach to increase β-cell replication and to improve islet graft function. It can potentially be used for human islet transplantation. Single-donor islet transplantation is a very attractive goal for clinical islet transplantation. The islet mass from single donors is often not adequate to restore normoglycemia, although diabetes could be reversed with islets isolated from a single-donor pancreas.11 Therefore, developing GPR119 agonist-based strategies to increase β-cell mass and to improve islet grafts function can potentially facilitate successful single-donor islet transplantation. Since human patients with still have some β-cell mass remaining at the onset of type 1 diabetes, GPR119 agonists may also be used to restore normoglycemia by stimulating β-cell replication and increasing β-cell mass.[2]

C₂₁H₂₄FN₇O₅S

505.52

505.154

C, 49.89; H, 4.79; F, 3.76; N, 19.40; O, 15.82; S, 6.34

733750-99-7

24939268

Light yellow to yellow solid powder

5.303

168.31

1

12

6

35

833

0

CC(C)C1=NOC(C2CCN(C3=NC=NC(NC4=CC=C(S(=O)(C)=O)C=C4F)=C3[N+]([O-])=O)CC2)=N1

DGBKNTVAKIFYNU-UHFFFAOYSA-N

InChI=1S/C21H24FN7O5S/c1-12(2)18-26-21(34-27-18)13-6-8-28(9-7-13)20-17(29(30)31)19(23-11-24-20)25-16-5-4-14(10-15(16)22)35(3,32)33/h4-5,10-13H,6-9H2,1-3H3,(H,23,24,25)

N-(2-fluoro-4-methylsulfonylphenyl)-5-nitro-6-[4-(3-propan-2-yl-1,2,4-oxadiazol-5-yl)piperidin-1-yl]pyrimidin-4-amine

AR-231453; AR 231453; AR231,453; 733750-99-7; AR 231,453; AR231,453; N-(2-Fluoro-4-(methylsulfonyl)phenyl)-6-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)-5-nitropyrimidin-4-amine; AR-231453; N-(2-fluoro-4-methylsulfonylphenyl)-5-nitro-6-[4-(3-propan-2-yl-1,2,4-oxadiazol-5-yl)piperidin-1-yl]pyrimidin-4-amine; CHEMBL461384; 07Z1P4981I; AR231,453; AR-231453; AR231,453

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)

DMSO: ~25 mg/mL (~49.5 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

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。