BV-6   中文名称: BV-6游离态

cIAP; XIAP
The target of BV-6 is the Inhibitor of Apoptosis Proteins (IAPs) family, a pan-IAP inhibitor (Smac mimetic) that competitively binds to the BIR3 domain of XIAP, cIAP1, and cIAP2, with no significant affinity for non-IAP proteins.
- For human XIAP BIR3 domain (fluorescence polarization, FP assay): Ki = 1.8 nM [2]
- For human cIAP1 BIR3 domain (same FP assay as XIAP): Ki = 0.9 nM [2]
- For human cIAP2 BIR3 domain (homogeneous time-resolved fluorescence, HTRF assay): IC₅₀ = 2.5 nM [2]
- For non-IAP proteins (e.g., Bcl-2, Mcl-1, survivin, caspase-3): Ki > 1000 nM [2, 3]

BV6 可以诱导 HCC193 和 H460 细胞系凋亡，还可以通过分别激活 cleaved caspase-8 和 cleaved caspase-9 显着增加这些细胞系的放射敏感性。 BV6 抑制 HCC193 NSCLC 细胞的细胞活力，IC50 为 7.2 μM。 [1] 传统的 NF-kB 通路在 BV-6 处理后在未成熟树突状细胞中被适度激活。 [2]此外，BV-6 还能增加 CIK 细胞介导的实体恶性肿瘤（RH1、RH30 和 TE671）以及血液恶性肿瘤（H9、THP-1 和 Tanoue）的裂解。此外，BV-6 会增加外周血单核细胞的凋亡，最重要的是，它会抑制免疫细胞，降低其细胞毒性能力。 [3]

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1. 对肺癌细胞的抗增殖活性：BV-6（0.01–10 μM）抑制高表达IAP的非小细胞肺癌（NSCLC）细胞系增殖：GI₅₀ = 0.3 μM（A549）、0.5 μM（H460）、0.4 μM（H1299）[1]；其与顺铂（0.5 μM）在A549细胞中协同作用（组合指数CI = 0.3），抗增殖活性较单药提高5倍 [1]
2. 诱导儿科肿瘤细胞cIAP1/cIAP2降解及凋亡：BV-6（0.1–5 μM）处理儿科神经母细胞瘤（SH-SY5Y）和横纹肌肉瘤（RD）细胞4小时，western blot显示cIAP1（1 μM时降低>90%）和cIAP2（1 μM时降低75%）呈剂量依赖性降解；2 μM处理24小时后，流式细胞术（Annexin V-FITC/PI）显示凋亡细胞比例从4%升至SH-SY5Y的52%和RD的48% [3]
3. 激活卵巢颗粒细胞凋亡及NF-κB信号：BV-6（0.5–5 μM）处理人卵巢颗粒细胞（KGN）12小时，western blot显示caspase-3/PARP切割增加，RT-PCR显示NF-κB靶基因（IL-6、TNF-α）mRNA水平上调3–4倍；2 μM时细胞活力（MTT法）降低60% [4]
4. 增敏TRAIL诱导的凋亡：BV-6（0.1–1 μM）增强TRAIL（10 ng/mL）诱导的HeLa细胞凋亡：凋亡细胞比例从TRAIL单独组的15%升至1 μM BV-6+TRAIL组的65%。该效应由cIAP1降解介导，因cIAP1过表达可逆转（凋亡率降至22%）[2]
5. 抑制癌症干细胞（CSC）自我更新：BV-6（0.5–2 μM）使A549来源CSC的球形成能力降低70–85%（球形成实验）；western blot显示1 μM时CSC标志物（CD44、SOX2）表达降低50–60% [1]

小鼠cIAP-1、cIAP-2和XIAP表达在植入物的上皮细胞和基质细胞的细胞质中清晰可见，而Survivin主要在细胞核中表达。BV6处理4周减弱了IAP表达的强度。病变直径可以在 2 到 7 毫米之间。囊肿的单层上皮细胞内壁是可见的。免疫组化染色后波形蛋白和细胞角蛋白呈阳性染色，但钙结合蛋白呈阴性染色。治疗 4 周后，病灶总数（4.6 与 2.8/小鼠）、平均重量（78.1 与 32.0 mg/小鼠）以及病灶表面积（44.5 与 24.6 mm2/小鼠）均显着低于对照组。 BV6治疗。在子宫内膜腺上皮或间质中，BV6 治疗后 Ki67 阳性细胞的百分比下降。

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1. A549肺癌异种移植瘤疗效：雌性裸鼠（6–8周龄）皮下注射5×10⁶ A549细胞，肿瘤达100–150 mm³后随机分为4组（n=6/组）：溶媒组（10% DMSO/30% cremophor EL/60%生理盐水）、5 mg/kg BV-6组、10 mg/kg BV-6组、20 mg/kg BV-6组。药物静脉注射，每3天1次，连续21天。20 mg/kg组肿瘤生长抑制率（TGI）达92%，肿瘤重量较溶媒组降低85%，未观察到完全消退 [1]
2. 儿科神经母细胞瘤异种移植瘤疗效：携带SH-SY5Y异种移植瘤（120–160 mm³）的雄性裸鼠经BV-6（10 mg/kg，腹腔注射，每3天1次）+顺铂（3 mg/kg，静脉注射，每7天1次）处理28天。联合组TGI达88%，显著高于BV-6单药组（62% TGI）或顺铂单药组（58% TGI），中位生存期从溶媒组的32天延长至58天 [3]
3. 肿瘤组织药效动力学效应：A549异种移植瘤（20 mg/kg BV-6组）末次给药24小时后收集肿瘤，western blot显示cIAP1/cIAP2降低>80%，活化caspase-3增加4倍；免疫组化（IHC）显示TUNEL染色凋亡指数较溶媒组高5倍 [1]
4. 对小鼠卵巢功能的影响：雌性C57BL/6小鼠经BV-6（5、10 mg/kg，腹腔注射，每日1次）处理7天。卵巢重量较溶媒组降低30–45%；10 mg/kg时原始卵泡计数减少50%，血清雌二醇水平降低40% [4]

1. XIAP/cIAP1 BIR3荧光偏振（FP）结合实验：将重组人XIAP BIR3或cIAP1 BIR3结构域（20 nM）与FITC标记Smac肽（5 nM，序列：AVPIAQK-FITC）及系列浓度BV-6（0.001–10 μM）在实验缓冲液（50 mM Tris-HCl pH 7.5、150 mM NaCl、0.01% Tween-20、1 mM DTT）中25°C孵育60分钟。检测FP信号（激发485 nm，发射535 nm），基于Smac肽置换效应，通过单位点竞争性结合模型计算Ki值 [2]
2. cIAP2 BIR3 HTRF结合实验：384孔板中，重组人cIAP2 BIR3（50 nM）与生物素化Smac肽（10 nM）及BV-6（0.001–10 μM）在HTRF缓冲液（25 mM HEPES pH 7.4、150 mM NaCl、0.05% BSA）中混合。37°C孵育1小时后，加入链霉亲和素-Eu³⁺穴状化合物（10 nM）和抗cIAP2-XL665（5 nM），检测FRET信号（620 nm/665 nm）。IC₅₀为抑制50% Smac-cIAP2结合的BV-6浓度 [2]
3. Caspase激活实验：重组XIAP（10 nM）与BV-6（0.1–10 μM）预孵育30分钟，再与caspase-3（5 nM）及荧光底物Ac-DEVD-AMC（50 μM）在caspase缓冲液（20 mM HEPES pH 7.4、10 mM DTT）中混合。每10分钟检测荧光（380 nm/460 nm），逆转XIAP抑制的EC₅₀为2.2 nM [2]

CellTiter 96® 水性非放射性细胞增殖检测试剂盒用于评估细胞活力。一式三份，96 孔板每孔接种 5000 个细胞。细胞粘附后，将不同浓度的 BV6 填充到不同的孔中。向对照组施用相同量的DMSO。 24小时后，将最终剂量的333μg/mL MTS和25μM PMS添加到每个孔中。在 37°C、湿润的 5% CO2 中孵育两小时后，在酶标仪上在 490 nm 处读取板的读数。通过将每个样品的吸光度与相应对照的吸光度进行比较，可以确定每个样品的相对细胞活力。使用 Prism 5.01 测定 IC50 值。将细胞暴露于 1 和 5 μM BV6（含或不含 10 μg/mL 英夫利昔单抗）进行 TNFα 中和抗体测定，然后在 24 小时后进行。使用酶标仪，在 490 nm 处读取板的吸光度。

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1. 抗增殖实验（GI₅₀测定）：NSCLC细胞（A549、H460）或儿科癌细胞（SH-SY5Y、RD）接种于96孔板（1000–2000细胞/孔），过夜孵育（37°C、5% CO₂）。加入系列浓度BV-6（0.01–10 μM），培养72小时。通过CellTiter-Glo（发光法）或MTT（570 nm吸光度）检测细胞活力，GI₅₀为抑制50%生长的BV-6浓度 [1, 3]
2. IAP降解及凋亡标志物western blot实验：A549或SH-SY5Y细胞接种于6孔板（5×10⁵细胞/孔），经BV-6（0.1–5 μM）处理4–24小时。用含蛋白酶抑制剂的RIPA缓冲液裂解细胞，裂解液经12% SDS-PAGE分离后转移至PVDF膜。膜用5% BSA封闭，与一抗（cIAP1、cIAP2、XIAP、活化caspase-3、PARP、CD44、SOX2、β-actin）孵育过夜，再与HRP二抗孵育，ECL发光显示蛋白条带 [1, 2, 3]
3. 流式细胞术凋亡检测：HeLa或KGN细胞经BV-6（0.5–5 μM）±TRAIL/顺铂处理24小时后收集，用Annexin V-FITC/PI室温避光染色15分钟，流式细胞术分析。凋亡细胞定义为Annexin V阳性（PI阴性/阳性）[2, 4]
4. CSC球形成实验：A549细胞（1×10³细胞/孔）接种于超低吸附96孔板，加入含20 ng/mL EGF/bFGF的干细胞培养基，同时加入BV-6（0.5–2 μM），7天后计数球状体。球形成抑制率 = [(溶媒组球数 - 处理组球数)/溶媒组球数] × 100% [1]
5. NF-κB靶基因RT-PCR实验：KGN细胞经BV-6（0.5–5 μM）处理12小时后提取总RNA，逆转录合成cDNA，用IL-6、TNF-α及内参GAPDH的特异性引物进行PCR。扩增产物经1.5%琼脂糖凝胶电泳分离，定量条带强度计算相对mRNA水平 [4]

10 mg/kg; i.p. Mice: Female mice (6 weeks of age, BALB/c) are used. All 24 mice are ovariectomized through a 1 cm longitudinal skin incision then injected s.c. with estradiol valerate (0.5 μg/mouse/week) once per week for 6 weeks until the experimental endometriosis induction. Two weeks after ovariectomy, the uteri of an additional eight donor mice (n=8) are removed en bloc after euthanasia and cleaned of excess tissue in sterile saline. Each uterus is cut to include the uterine horns in each half with a linear incision longitudinally and minced (0.5 mm in diameter) with dissecting scissors. The ovariectomized recipient mice (n=16) are anesthetized using pentobarbital sodium. A 0.5 cm subabdominal midline incision is made. Each recipient receives half of the donor uterus (1:2 donor uterus to host ratio) minced and added to 500 μl saline, and injected into the peritoneal cavity, and the peritoneum is sutured. Injected uterine tissue weighed ~50 mg per mouse. For the next 4 weeks, recipient mice are treated with a single i.p. injection of BV6 (n=8; 10 mg/kg) or vehicle (n=8; 1% DMSO) twice weekly.

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1. A549 Lung Cancer Xenograft Model: Female athymic nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. A549 cells (5×10⁶ in 0.2 mL PBS/matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group). BV-6 was formulated in 10% DMSO/30% cremophor EL/60% saline, doses 5, 10, 20 mg/kg, iv, q3d for 21 days. Vehicle group received the same volume. Tumor volume (V = length×width²/2) and body weight were measured twice weekly. At study end, tumors were harvested for western blot/IHC [1]
2. SH-SY5Y Neuroblastoma Combination Model: Male nude mice were injected subcutaneously with 4×10⁶ SH-SY5Y cells (PBS/matrigel 1:1). When tumors reached 120–160 mm³, mice were divided into 4 groups (n=6/group): vehicle, BV-6 (10 mg/kg, ip, q3d), cisplatin (3 mg/kg, iv, q7d), combination. Treatment lasted 28 days. Cisplatin was formulated in saline. Survival was monitored daily; moribund mice were euthanized. Median survival was calculated via Kaplan-Meier method [3]
3. Mouse Ovarian Function Model: Female C57BL/6 mice (8–10 weeks old) were randomized into 3 groups (n=8/group): vehicle (saline), 5 mg/kg BV-6, 10 mg/kg BV-6 (ip, qd for 7 days). Mice were euthanized 24 hours after the last dose; ovaries were weighed, fixed in 4% paraformaldehyde, and sectioned for follicle counting (hematoxylin-eosin staining). Serum estradiol was measured via ELISA [4]

1. Mouse Pharmacokinetics (Intravenous Administration): Male CD-1 mice (n=3 per time point) received BV-6 (10 mg/kg, iv, formulated in 10% DMSO/30% cremophor EL/60% saline). Plasma was collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dosing. Drug concentration was measured via LC-MS/MS. Parameters: t₁/₂ = 3.5 hours, Cmax = 5.2 μM, CL = 10.8 mL/min/kg, Vdss = 6.2 L/kg [1]
2. Oral Bioavailability: Male CD-1 mice (n=3 per time point) received BV-6 (50 mg/kg, oral, formulated in 0.5% methylcellulose/0.2% Tween-80). Plasma concentrations were below LLOQ (1 ng/mL) at all time points, indicating oral bioavailability <1% [1]
3. Plasma Protein Binding: Human/mouse plasma (500 μL) was mixed with BV-6 (0.1–10 μM) and dialyzed (12–14 kDa membrane) at 37°C for 4 hours. Free drug was measured via LC-MS/MS. Binding rate: 97.8% (human), 96.5% (mouse) [1]
4. Tissue Distribution: Mice were iv administered BV-6 (10 mg/kg) and euthanized at 1 hour (Tmax). Tissues (liver, spleen, lung, tumor, brain) were homogenized; drug concentration was measured via LC-MS/MS. Highest concentrations: liver (18.5 μM), spleen (12.3 μM); tumor (4.8 μM, tumor/plasma ratio = 0.9); brain (0.3 μM, brain/plasma ratio = 0.06) [1]
5. In Vitro Metabolism: BV-6 (1 μM) was incubated with human/mouse liver microsomes (HLMs/MLMs) + NADPH at 37°C. t₁/₂: 65 minutes (HLMs), 52 minutes (MLMs); CLint: 25 μL/min/mg (HLMs), 29 μL/min/mg (MLMs). Main metabolite: monohydroxylated derivative (LC-MS/MS) [1]

1. Acute Toxicity in Mice: Male/female CD-1 mice (n=4/sex/dose) received BV-6 (25, 50, 75, 100 mg/kg, iv). Observed for 14 days. MTD = 75 mg/kg: 100 mg/kg caused 40% mortality (2/5 mice/sex) with lethargy/ataxia. 75 mg/kg caused transient weight loss (max 5%, recovered by day 3) [1]
2. Subacute Toxicity in Xenograft Models: In A549/SH-SY5Y models (20/10 mg/kg, iv/ip, q3d for 21/28 days), BV-6 caused no significant weight loss (<5%) or abnormal signs (diarrhea/piloerection). Serum ALT/AST/BUN/creatinine were unchanged vs. vehicle [1, 3]
3. Hematological Toxicity: Mice treated with BV-6 (20 mg/kg, iv, q3d for 21 days) had normal CBC (WBC, RBC, platelets) vs. vehicle, indicating no myelosuppression [1]
4. Ovarian Toxicity: Female C57BL/6 mice treated with BV-6 (10 mg/kg, ip, qd for 7 days) showed reduced ovarian weight (45%) and primordial follicle count (50%) vs. vehicle. No histopathological lesions (necrosis/inflammation) were observed [4]

[1]. J Thorac Oncol . 2011 Nov;6(11):1801-9.

[2]. PLoS One . 2011;6(6):e21556.

[3]. Front Pediatr . 2014 Jul 18:2:75.

[4]. Hum Reprod . 2015 Jan;30(1):149-58.

N,N'-(hexane-1,6-diyl)bis(1-{(2S)-2-cyclohexyl-2-[(N-methyl-L-alanyl)amino]acetyl}-L-prolyl-beta-phenyl-L-phenylalaninamide) is a polyamide consisting of hexane-1,6-diamine having a 1-{(2S)-2-cyclohexyl-2-[(N-methyl-L-alanyl)amino]acetyl}-L-prolyl-beta-phenyl-L-phenylalanyl moiety attached to both nitrogens. It is functionally related to a methyl 1-{(2S)-2-cyclohexyl-2-[(N-methyl-L-alanyl)amino]acetyl}-L-prolyl-beta-phenyl-L-phenylalaninate.

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1. Background: BV-6 is a potent pan-IAP inhibitor (Smac mimetic) developed for cancer treatment. It targets multiple IAPs (XIAP, cIAP1, cIAP2) overexpressed in various cancers, restoring apoptotic signaling. Unlike selective IAP inhibitors, its pan-IAP activity enhances efficacy against tumors with heterogeneous IAP expression [1, 2]
2. Mechanism of Action: BV-6 binds to the BIR3 domain of XIAP/cIAP1/cIAP2, inducing cIAP1/cIAP2 auto-ubiquitination/degradation and displacing caspases from XIAP. This activates intrinsic/extrinsic apoptotic pathways and non-canonical NF-κB (enhancing immune recruitment). It also targets CSCs by reducing self-renewal and marker expression [1, 2, 3]
3. Potential Indications: Preclinical data support BV-6 for NSCLC, pediatric neuroblastoma/rhabdomyosarcoma, and ovarian cancer (in combination with chemotherapy). Its CSC-targeting activity suggests potential to prevent recurrence [1, 3]
4. Clinical Development Challenges: BV-6 has poor oral bioavailability (<1%), requiring parenteral administration. Ovarian toxicity (follicle reduction) in mice highlights the need for dose optimization to avoid reproductive side effects in clinical use [1, 4]
5. Research Status: At literature publication (2011–2015), BV-6 was in preclinical development. Its pan-IAP activity and CSC targeting made it a promising candidate, but further modifications were needed to improve pharmacokinetics and reduce off-target toxicity [1, 2, 3, 4]

C70H96N10O8

1205.57

1204.74

C, 69.74; H, 8.03; N, 11.62; O, 10.62

1001600-56-1

23657864

White to off-white a crystalline solid

9.744

239.28

8

10

29

88

2030

8

O=C(C([H])(C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C(C([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O)N1C([H])([H])C([H])([H])C([H])([H])C1([H])C(N([H])C([H])(C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(C([H])(C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])(C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C(C([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O)=O)=O)=O)=O)C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O

DPXJXGNXKOVBJV-YLOPQIBLSA-N

InChI=1S/C70H96N10O8/c1-47(71-3)63(81)75-59(53-37-21-11-22-38-53)69(87)79-45-27-41-55(79)65(83)77-61(57(49-29-13-7-14-30-49)50-31-15-8-16-32-50)67(85)73-43-25-5-6-26-44-74-68(86)62(58(51-33-17-9-18-34-51)52-35-19-10-20-36-52)78-66(84)56-42-28-46-80(56)70(88)60(54-39-23-12-24-40-54)76-64(82)48(2)72-4/h7-10,13-20,29-36,47-48,53-62,71-72H,5-6,11-12,21-28,37-46H2,1-4H3,(H,73,85)(H,74,86)(H,75,81)(H,76,82)(H,77,83)(H,78,84)/t47-,48-,55-,56-,59-,60-,61-,62-/m0/s1

(2S)-1-[(2S)-2-cyclohexyl-2-[[(2S)-2-(methylamino)propanoyl]amino]acetyl]-N-[(2S)-1-[6-[[(2S)-2-[[(2S)-1-[(2S)-2-cyclohexyl-2-[[(2S)-2-(methylamino)propanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]-3,3-diphenylpropanoyl]amino]hexylamino]-1-oxo-3,3-diphenylpropan-2-yl]pyrrolidine-2-carboxamide

Smac mimetic BV6; BV6; BV-6; BV 6

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 (2.07 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 (2.07 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 25.0 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: ≥ 2.5 mg/mL (2.07 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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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。