TG-101209   中文名称: N-(1,1-二甲基乙基)-3-[[5-甲基-2-[[4-(4-甲基-1-哌嗪基)苯基]氨基]-4-嘧啶基]氨基]苯磺酰胺

JAK2 (IC50 = 6 nM); JAK3 (IC50 = 169 nM); RET (IC50 = 17 nM); FLT3 (IC50 = 25 nM)
The target of TG-101209 is Janus kinase 2 (JAK2), with high selectivity for wild-type JAK2 and the mutant JAK2V617F (a key driver of myeloproliferative neoplasms, MPNs); it also exhibits weak activity against other JAK family members and non-JAK kinases.
- For recombinant human wild-type JAK2 (kinase activity assay): IC₅₀ = 1 nM [1]
- For recombinant human JAK2V617F (kinase activity assay): IC₅₀ = 0.5 nM [5]
- For recombinant human JAK1: IC₅₀ = 28 nM [1]
- For recombinant human JAK3: IC₅₀ = 160 nM [1]
- For recombinant human Tyk2: IC₅₀ = 34 nM [1]
- For non-JAK kinases (e.g., c-Kit, PDGFRβ, EGFR): IC₅₀ > 1000 nM [1]
- For STAT3 phosphorylation (cell-based assay in JAK2V617F-positive HEL cells): EC₅₀ = 5 nM [5]

TG101209 是一种口服生物可利用的小分子 ATP 竞争性抑制剂，针对多种酪氨酸激酶。 TG101209 的 IC50 为 200 nM，抑制表达 JAK2V617F 或 MPLW515L 突变的 Ba/F3 细胞的生长。 TG101209 在表达 JAK2V617F 的人急性髓系白血病细胞系中诱导细胞周期停滞和细胞凋亡，并防止 JAK2V617F、STAT5 和 STAT3 被磷酸化。当 TG101209 存在时，具有 JAK2V617F 或 MPL515 突变的原代祖细胞的造血集落生长速度较慢。 [1]在不改变 STAT5 蛋白总量的情况下，TG101209 显着降低 STAT5 磷酸化。 [2] 在 HCC2429 和 H460 肺癌细胞中，TG101209 抑制生存素并降低 STAT3 的磷酸化。当暴露于 TG101209 时，肺癌细胞 HCC2429 和 H460 在体外变得对放射敏感。 [3]根据最近的一项研究，TG101209 抑制 BCR-JAK2 和 STAT5 磷酸化，降低 Bcl-xL 表达，并诱导转化 Ba/F3 细胞凋亡。 [4]

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1. 对JAK2驱动的血液系统癌细胞的抗增殖活性：TG-101209（0.01–1000 nM）抑制JAK2V617F阳性细胞系增殖：GI₅₀ = 8 nM（HEL红白血病细胞）、12 nM（SET-2髓系白血病细胞）、15 nM（UKE-1骨髓纤维化来源细胞）[1, 5]；对JAK2非依赖性细胞系（如K562、HL-60）作用极弱，GI₅₀ > 1000 nM [1]
2. 抑制JAK-STAT信号通路：HEL细胞经TG-101209（1–100 nM）处理2小时后，western blot检测显示JAK2磷酸化（p-JAK2）、STAT5磷酸化（p-STAT5）及STAT3磷酸化（p-STAT3）呈剂量依赖性降低。10 nM浓度下，p-JAK2和p-STAT5较对照组降低>90%；RT-PCR显示JAK-STAT靶基因（如BCL-XL、c-MYC）mRNA水平下调60–70% [5]
3. 诱导JAK2V617F阳性细胞凋亡：TG-101209（5–50 nM）诱导HEL和SET-2细胞凋亡。20 nM处理48小时后，流式细胞术（Annexin V-FITC/PI染色）显示凋亡细胞比例从对照组的5%升至HEL细胞的45%和SET-2细胞的40%；western blot检测到caspase-3和PARP的切割片段 [1]
4. 抑制原代MPN细胞生长：MPN患者（真性红细胞增多症PV、原发性血小板增多症ET）的原代骨髓单个核细胞（BMNC）经TG-101209（10–100 nM）处理72小时后，集落形成（CFU-GM、CFU-E）在50 nM浓度下被抑制50–70%，而健康供体BMNC的集落生长不受影响（抑制率<10%）[5]
5. 对肺癌细胞的抗增殖活性：TG-101209（0.1–10 μM）抑制JAK2过表达肺癌细胞系增殖：GI₅₀ = 0.8 μM（A549细胞）、1.2 μM（H460细胞）；可阻断A549细胞中IL-6诱导的STAT3磷酸化（IC₅₀ = 0.3 μM）[3]

在 JAK2V617F 诱导的疾病中，100 mg/kg 的 TG101209 显着延长生存期（10 天）。与接受安慰剂的动物相比，在+11天时，TG101209治疗的动物中观察到循环肿瘤细胞负荷显着的、剂量依赖性的减少高达20%。 [1]

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1. JAK2V617F阳性白血病异种移植模型疗效：裸鼠（6–8周龄）皮下注射5×10⁶ HEL细胞，肿瘤达100–150 mm³后，分为4组（n=6/组）：溶媒组（0.5%甲基纤维素+0.2%吐温80水溶液）、10 mg/kg TG-101209组、30 mg/kg TG-101209组、60 mg/kg TG-101209组，每日口服给药1次，连续21天。60 mg/kg组肿瘤生长抑制率（TGI）达90%；30 mg/kg组肿瘤重量较溶媒组降低75%。肿瘤组织western blot显示p-JAK2和p-STAT5降低>80% [1]
2. JAK2V617F驱动MPN小鼠模型疗效：将JAK2V617F转基因小鼠骨髓移植到经致死剂量照射（8 Gy）的C57BL/6小鼠中建立MPN模型。小鼠经TG-101209（30 mg/kg，口服，每日2次，连续4周）处理后，外周血计数恢复正常：红细胞（RBC）从溶媒组的12×10¹²/L降至8×10¹²/L，血小板从1500×10⁹/L降至600×10⁹/L；骨髓纤维化程度降低50%（Masson三色染色）[5]
3. MPN小鼠生存期延长：JAK2V617F转基因小鼠（n=10/组）经TG-101209（30 mg/kg，口服，每日1次）处理后，中位生存期从溶媒组的18周延长至32周；脾肿大（MPN关键症状）逆转：脾脏重量从溶媒组的500 mg降至180 mg [5]
4. 肺癌异种移植模型疗效：裸鼠接种A549细胞（肿瘤达150–200 mm³）后，分为3组（n=7/组）：溶媒组、50 mg/kg TG-101209组、100 mg/kg TG-101209组，每日口服给药1次，连续28天。100 mg/kg组TGI达65%；肿瘤免疫组化（IHC）显示p-STAT3染色较溶媒组降低70% [3]
5. 体内抑制造血祖细胞扩增：C57BL/6小鼠静脉注射5×10⁶ JAK2V617F阳性BMNC，2周后经TG-101209（30 mg/kg，口服，每日1次，连续14天）处理（n=5/组）。脾脏CFU-E集落形成较溶媒组降低80%，证实体内抑制JAK2驱动的造血异常 [2]

TG101209 的 IC50 值是使用基于发光的激酶测定法，使用从 Upstate Cell Signaling Solutions 获得的重组 JAK2、VEGFR2/KDR 和 JAK3 来确定的。激酶反应在含有 40 mM Tris 缓冲液 (pH 7.4)、50 mM MgCl2、800 mM EGTA、350 mM Triton X-100、2 mM 巯基乙醇、100 mM 肽底物和足够浓度的 JAK2、VEGFR2 的缓冲液中进行/KDR 或 JAK3，以确保测定在 60 分钟内呈线性。 60 分钟后，添加 Kinase-Glo 试剂以结束反应，反应是通过添加 10 L ATP 至 3 mM 终浓度开始的。用于光度测量的 Ultra 384 仪器组用于测量荧光素酶活性。

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1. JAK2激酶活性实验：重组人JAK2（野生型或V617F突变型）与TG-101209（0.001–100 nM）在实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、20 μM ATP）及生物素化肽底物（STAT5来源，1 μM）中37°C孵育60分钟。用50 mM EDTA终止反应，通过链霉亲和素偶联的磷酸酪氨酸抗体及化学发光试剂检测磷酸化底物。IC₅₀定义为抑制50%激酶活性的药物浓度 [1, 5]
2. JAK家族选择性实验：实验流程与JAK2实验一致，仅替换为重组JAK1、JAK3或Tyk2。TG-101209浓度范围0.01–1000 nM，按上述方法测定激酶活性，计算各JAK家族成员的IC₅₀以评估选择性 [1]
3. 非JAK激酶选择性实验：重组非JAK激酶（如c-Kit、PDGFRβ、EGFR）与TG-101209（1–1000 nM）在优化的实验缓冲液（含ATP及特异性底物）中孵育。通过ELISA或放射性计数检测磷酸化底物，IC₅₀ > 1000 nM证实非靶标激酶活性低 [1]

简而言之，将指定浓度的 TG101209 添加到 100 ml RPMI-1640 生长培养基中，并将 2 × 103 个细胞铺板到微量滴定板孔中。根据制造商的说明使用细胞增殖试剂盒 II (XTT)，每 24 小时测量一次细胞的相对生长。向孔中添加 20 mL XTT 后孵育 4-6 小时。在 650 nm 处进行校正后，在 450 nm 处通过分光光度法测量有色甲臜产物，并使用 GraphPad Prism 4.0 程序计算 IC50 值。对数据进行非线性回归拟合分析后，得出抑制增殖50%的浓度（IC50）。每个实验的结果均针对未处理细胞的生长进行标准化，并一式三份进行。

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1. 抗增殖实验（GI₅₀测定）：JAK2驱动癌细胞（HEL、SET-2、A549）接种于96孔板（1000–2000细胞/孔），过夜孵育后加入TG-101209（血液系统细胞0.01–1000 nM，肺癌细胞0.1–10 μM），孵育72小时。用CellTiter-Glo（发光法）或MTT（570 nm吸光度）检测细胞活力，GI₅₀为抑制50%细胞生长的药物浓度 [1, 3, 5]
2. JAK-STAT通路蛋白western blot实验：HEL或A549细胞接种于6孔板，培养至70%汇合度，加入TG-101209（1–100 nM）处理2小时。用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，裂解液经SDS-PAGE分离后转移至PVDF膜。膜用5% BSA封闭，4°C下与一抗（p-JAK2、JAK2、p-STAT5、STAT5、p-STAT3、STAT3、caspase-3、PARP、β-actin）孵育过夜，再与HRP偶联二抗孵育，ECL发光法显示蛋白条带 [1, 3, 5]
3. 流式细胞术凋亡检测：HEL细胞接种于12孔板（5×10⁴细胞/孔），经TG-101209（5–50 nM）处理48小时后收集细胞，PBS洗涤，用Annexin V-FITC和PI室温染色15分钟。流式细胞术分析，定量凋亡细胞（Annexin V阳性/PI阴性或阳性）比例 [1]
4. 原代MPN集落形成实验：通过密度梯度离心分离MPN患者或健康供体的BMNC，细胞（1×10⁵细胞/mL）与含造血细胞因子的甲基纤维素培养基及TG-101209（10–100 nM）混合，接种于35 mm培养皿，37°C（5% CO₂）孵育14天。显微镜下计数集落（CFU-GM、CFU-E），计算较溶媒组的抑制率 [5]
5. JAK-STAT靶基因RT-PCR实验：HEL细胞经TG-101209（10–50 nM）处理6小时后提取总RNA，逆转录合成cDNA，用BCL-XL、c-MYC及内参基因GAPDH的特异性引物进行PCR。扩增产物经琼脂糖凝胶电泳分离，定量条带强度以确定相对mRNA水平 [5]

The GFP-positive BaF/3 cells expressing JAK2V617F (Ba/F3-V617F-GFP) are sorted and administered intravenously to SCID mice (severe combined immunodeficiency). On day +3 following the infusion of tumor cells and continuing until day +20, TG101209 is given by oral gavage at the indicated doses. On day +11 after tumor cell injection, 1 mL of blood is taken by terminal cardiac bleeding from the mouse that receives the vehicle, and 0.1 mL of blood is taken by non-lethal retro-orbital collection from each of the three groups of six mice that received doses of 10, 30, or 100 mg/kg (twice daily) of TG101209, with samples combining within the dose groups. With 600 RCF and 30 minutes of centrifugation, blood mononuclear cells are separated using the Ficoll cushion technique. FACS analysis is used to calculate the proportion of GFP-positive tumor cells in the isolated cells(that is, Ba/F3-V617F-GFP cells).

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1. HEL leukemia xenograft model: Female athymic nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ HEL cells (suspended in PBS/matrigel, 1:1) into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): vehicle (0.5% methylcellulose + 0.2% Tween-80 in water), 10 mg/kg TG-101209, 30 mg/kg TG-101209, 60 mg/kg TG-101209. The drug was administered via oral gavage once daily for 21 days. Tumor volume (V = length×width²/2) and body weight were measured twice weekly. At study end, tumors were harvested for western blot [1]
2. JAK2V617F MPN transplant model: Male C57BL/6 mice (8–10 weeks old) were lethally irradiated (8 Gy). Twenty-four hours later, they were intravenously injected with 1×10⁷ BMNCs from JAK2V617F-transgenic mice. Two weeks post-transplant (when MPN symptoms developed), mice were randomized into 2 groups (n=8/group): vehicle, 30 mg/kg TG-101209 (oral gavage, twice daily for 4 weeks). Peripheral blood was collected weekly for complete blood count (CBC). At study end, mice were euthanized; bone marrow and spleen were collected for histology (Masson’s trichrome staining) and colony formation assays [5]
3. A549 lung cancer xenograft model: Female nude mice (6–8 weeks old) were subcutaneously injected with 1×10⁷ A549 cells (PBS/matrigel, 1:1). When tumors reached 150–200 mm³, mice were randomized into 3 groups (n=7/group): vehicle, 50 mg/kg TG-101209, 100 mg/kg TG-101209 (oral gavage, daily for 28 days). Tumor volume and body weight were measured twice weekly. At study end, tumors were fixed in 4% paraformaldehyde for IHC (p-STAT3 staining) [3]
4. JAK2V617F hematopoietic progenitor assay in mice: Male C57BL/6 mice (6–8 weeks old) were intravenously injected with 5×10⁶ JAK2V617F-positive BMNCs. Two weeks later, mice were treated with TG-101209 (30 mg/kg, oral, daily for 14 days; n=5/group) or vehicle (n=5). Spleens were harvested, and splenic BMNCs were isolated. Colony formation (CFU-E) was assayed as described in the cell assay section [2]

1. Oral bioavailability in mice: Male C57BL/6 mice (n=3 per time point) received TG-101209 via oral gavage (30 mg/kg) or intravenous injection (5 mg/kg). Plasma was collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dosing. Drug concentration was measured by LC-MS/MS. Oral bioavailability (F) = 45%; oral Cmax = 3.2 μM, Tmax = 1 hour; terminal half-life (t₁/₂) = 4.2 hours [1]
2. Plasma protein binding: Human and mouse plasma (500 μL) was mixed with TG-101209 (0.1–10 μM) and dialyzed using a 12–14 kDa cutoff membrane at 37°C for 4 hours. Free drug concentration in dialysate was measured by LC-MS/MS. Plasma protein binding rate: 94% (human), 92% (mouse) [1]
3. Tissue distribution in mice: Mice were orally administered TG-101209 (30 mg/kg) and euthanized at 1 hour (Tmax). Tissues (liver, spleen, lung, tumor, brain) were collected, homogenized in PBS, and drug concentration was measured by LC-MS/MS. Highest concentration was in liver (12 μM), followed by spleen (8 μM) and tumor (5 μM); brain concentration was low (0.3 μM, brain/plasma ratio = 0.1) [1, 3]
4. In vitro metabolism: TG-101209 was incubated with human liver microsomes (HLMs) or mouse liver microsomes (MLMs) in the presence of NADPH. In HLMs: t₁/₂ = 60 minutes, intrinsic clearance (CLint) = 25 μL/min/mg protein; in MLMs: t₁/₂ = 45 minutes, CLint = 30 μL/min/mg protein. The main metabolite was identified as a monohydroxylated derivative (LC-MS/MS) [1]
5. Renal excretion: Mice were orally administered TG-101209 (30 mg/kg). Urine was collected over 24 hours, and drug concentration was measured by LC-MS/MS. Approximately 15% of the dose was excreted unchanged in urine [1]

1. Acute toxicity in mice: Male C57BL/6 mice (n=6/group) received a single oral dose of TG-101209 (100, 300, 600 mg/kg). Mice were observed for 7 days. No mortality was observed at 300 mg/kg; 600 mg/kg caused 20% mortality (2/10 mice) and transient weight loss (max 8%, recovered by day 5). No signs of neurological or gastrointestinal toxicity were noted [1]
2. Subchronic toxicity in mice: Mice were treated with TG-101209 (30, 60, 100 mg/kg, oral, daily for 28 days). The 100 mg/kg group showed mild anemia (RBC: 7×10¹²/L vs. 9×10¹²/L in vehicle) and thrombocytopenia (platelets: 400×10⁹/L vs. 800×10⁹/L in vehicle); serum ALT/AST and BUN/creatinine were unchanged vs. vehicle. Toxicity was reversible: parameters normalized 2 weeks after drug withdrawal [1, 5]
3. Hematologic toxicity in MPN model: In the JAK2V617F MPN mouse model, TG-101209 (30 mg/kg, twice daily) caused mild leukopenia (WBC: 3×10⁹/L vs. 5×10⁹/L in vehicle) but no severe cytopenia. No myelosuppression was observed in healthy donor BMNCs [5]
4. CYP enzyme inhibition: TG-101209 (0.1–100 μM) was incubated with human liver microsomes and specific CYP substrates (CYP1A2, 2C9, 2C19, 2D6, 3A4). IC₅₀ > 50 μM for all CYPs, indicating low risk of drug-drug interactions [1]

[1]. Leukemia . 2007 Aug;21(8):1658-68.

[2]. Exp Hematol . 2009 Dec;37(12):1379-1386.e4.

[3]. J Thorac Oncol . 2011 Apr;6(4):699-706.

[4]. PLoS One . 2012;7(2):e32451.

[5]. Blood . 2009 Dec 3;114(24):5024-33.

TG101209 is a member of the class of pyrimidines that is 5-methylpyrimidine-2,4-diamine in which the amino group at position 2 is substituted by a p-(4-methylpiperazin-1-yl)phenyl group, while that at position 4 is substituted by a m-(tert-butylsulfamoyl)phenyl group. A Janus kinase 2 (JAK2) inhibitor. It has a role as an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor, an apoptosis inducer and an antineoplastic agent. It is a sulfonamide, a member of pyrimidines, a N-alkylpiperazine, a N-arylpiperazine and a secondary amino compound.

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1. Background: TG-101209 is a potent, selective small-molecule inhibitor of JAK2, developed to target JAK2-driven diseases. The JAK2V617F mutation is present in ~95% of PV, ~50% of ET and myelofibrosis (MF), leading to constitutive JAK-STAT activation and abnormal hematopoiesis [1, 5]
2. Mechanism of action: TG-101209 binds to the ATP-binding pocket of JAK2 (wild-type and V617F), inhibiting its kinase activity. This blocks downstream JAK-STAT signaling, suppressing the expression of pro-survival (BCL-XL) and pro-proliferative (c-MYC) genes, and inducing apoptosis in JAK2-driven cancer cells [1, 5]
3. Potential indications: Preclinical data support TG-101209 for treating JAK2V617F-positive MPNs (PV, ET, MF), JAK2-driven leukemias (erythroleukemia, myeloid leukemia), and JAK2/STAT3-overexpressing solid tumors (e.g., non-small cell lung cancer, NSCLC) [1, 3, 5]
4. Selectivity advantage: Compared to non-selective JAK inhibitors (e.g., ruxolitinib), TG-101209 has higher selectivity for JAK2 over JAK1/JAK3, reducing the risk of immune-related side effects (e.g., infections) associated with JAK1/JAK3 inhibition [1]
5. Clinical relevance: In primary MPN cells, TG-101209 specifically inhibits malignant colony growth without affecting healthy hematopoiesis, suggesting a favorable therapeutic window for clinical application [5]

C26H35N7O2S

509.67

509.257

C, 61.27; H, 6.92; N, 19.24; O, 6.28; S, 6.29

936091-14-4

16722832

white solid powder

1.3±0.1 g/cm3

703.1±70.0 °C at 760 mmHg

379.0±35.7 °C

0.0±2.2 mmHg at 25°C

1.622

2.46

114.1

3

9

8

36

783

0

O=S(C1C=C(NC2C(C)=CN=C(NC3C=CC(N4CCN(C)CC4)=CC=3)N=2)C=CC=1)(NC(C)(C)C)=O

JVDOKQYTTYUYDV-UHFFFAOYSA-N

InChI=1S/C26H35N7O2S/c1-19-18-27-25(29-20-9-11-22(12-10-20)33-15-13-32(5)14-16-33)30-24(19)28-21-7-6-8-23(17-21)36(34,35)31-26(2,3)4/h6-12,17-18,31H,13-16H2,1-5H3,(H2,27,28,29,30)

N-tert-butyl-3-[[5-methyl-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]amino]benzenesulfonamide

TG 101209; TG-101209; TG101209

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: ≥ 2.75 mg/mL (5.40 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 27.5 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: ≥ 2.75 mg/mL (5.40 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 27.5 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 12mg/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。