Brefeldin A (BFA)   中文名称: 布雷非德菌素 A

Natural product; CRISPR/Cas9; HSV-1; Arf-GEFs

用布雷菲德菌素 A (BFA) 处理 15 或 40 小时后，内质网 (ER) 显着肿胀并移动到正常肾 (NRK) 细胞的外周。长期 Brefeldin A 治疗会显着破坏肌动蛋白和 MT 细胞骨架 [1]。 Brefeldin A 和 ADPR 缀合物介导 BARS 的 ADP 核糖基化。当使用从用 BFA 处理的 CD38+ HeLa 细胞获得的细胞创建时，条形图显示了 BAC 结合 [3]。 Brefeldin A 减少 3D 和 2D 培养物中的 MDA-MB-231 集落形成，促进 MDA-MB-231 乳腺癌细胞中身份无关的细胞死亡，并阻断 MDA-MB 迁移和 MMP 9（基质金属肽酶 9）活性 - 231 [2]。

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肿瘤干细胞（Cancer stem cells, CSCs）是肿瘤细胞或已建立的癌细胞系的一个子集，可以在体内启动和维持肿瘤的生长。癌症干细胞可以在无血清的悬浮培养物中富集，在几天到几周内形成肿瘤球。Brefeldin A （BFA）是一种在真核细胞中诱导内质网（ER）应激的真菌毒素。我们发现，与粘附培养物相比，亚微克/毫升浓度的BFA在MDA-MB-231悬浮培养物（EC50: 0.016µg/mL）中优先诱导细胞死亡。BFA还能有效抑制MDA-MB-231细胞的克隆活性、迁移和基质金属蛋白酶-9 （MMP-9）活性。Western blotting分析表明，BFA的作用可能通过下调乳腺CSC标志物CD44和抗凋亡蛋白Bcl-2、Mcl-1，逆转上皮-间质转化介导。此外，BFA对悬浮的MDA-MB-468细胞也表现出选择性的细胞毒性，并抑制T47D和MDA-MB-453细胞的肿瘤球形成，表明BFA可能对各种表型的乳腺癌细胞有效。[2]

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糖蛋白D （gD-1）是1型单纯疱疹病毒（HSV-1）的重要病毒粒子包膜成分，通常被转运到感染细胞的质膜上。本研究在体外培养的HSV-1感染的人成纤维细胞中，加入1微克/毫升布雷菲尔丁A (Brefeldin A， BFA)，吸附病毒12 h后，抑制gD-1在细胞内的转运。免疫荧光和共聚焦显微镜显示gD-1向质膜的运输被取消，gD-1在核旁积聚，微管纤维排列混乱。BFA影响的解除超过60分钟导致不完全运输，但增加了质膜和靠近细胞核的高尔基样区域中gD-1的积累。微管蛋白模式在去除BFA后6小时基本恢复正常。bfa处理后9小时释放的感染性HSV-1颗粒未完全恢复。结果表明，BFA的作用不是完全可逆的，并引起一种涉及微管蛋白结构的细胞毒性影响。［７］

M-BFA(BFA encapsulated in mixed nanomicelles based on TPGS and F127 copolymers))的体内抗肿瘤效果 [8]
鉴于M-BFA具有突出的体外细胞毒性和较高的体内肿瘤蓄积性，我们利用HepG2荷瘤异种移植模型进一步研究了M-BFA的抗肿瘤作用。将小鼠分为三组，每天静脉注射PBS、M-BFA 5 mg/kg和M-BFA 10 mg/kg，连续14 d。如图7A、C所示，M-BFA 10 mg/kg组具有较强的抗肿瘤作用，可显著延缓肿瘤进展，而M-BFA 5 mg/kg组无明显抑制作用。M-BFA 10 mg/kg组肿瘤生长抑制率（TGI %）约为42.08%±3.29%，是M-BFA 5 mg/kg组的2倍。在整个实验过程中，三组均造成最小的动物体重减轻（图7B），表明毒性较低。苏木精和伊红染色（H&E）分析显示M-BFA表现出广泛的肿瘤坏死（图7D）。如图7D所示，给药M-BFA后，肿瘤细胞呈片状坏死。坏死灶呈粉红色，甚至部分坏死肿瘤组织溶解，形成空腔（红色箭头）。坏死灶内可见较多中性粒细胞浸润（绿箭头）。肿瘤细胞核质比大，少数细胞出现有丝分裂（黄箭头）。

先前对Brefeldin A （BFA）作用的研究主要集中在er -高尔基膜运输的动力学上，主要是在相对较短的药物治疗后。我们现在已经分析了长时间BFA处理对整体细胞形态、常驻和循环高尔基蛋白行为以及微管和肌动蛋白细胞骨架组织的影响。长时间（15小时或40小时）用BFA处理正常大鼠肾（NRK）细胞，引起内质网（ER）的剧烈肿胀，并将其定位转移到细胞周围。高尔基复合体被分解，高尔基蛋白在部分不同的区室中重新分布并持续存在。长时间的BFA治疗导致MT和肌动蛋白细胞骨架的明显破坏。外周MT缺失，微管蛋白染色集中在微管组织中心（MTOC）发出的短星状MT。肌动蛋白应力纤维大部分缺失，肌动蛋白染色集中在核周区域。在这个区域内，肌动蛋白的定位与膜转运因子p115的定位重叠。BFA对高尔基体结构、MT和肌动蛋白组织的影响表现出相同的阈值——在BFA处理30分钟和15小时后，这些影响都可以部分逆转，但在与药物孵化40小时后，这些影响是不可逆的。所观察到的效应不是由参与凋亡现象的信号通路或内质网应激反应通路诱导的。这些结果表明，BFA抑制了调节MT和肌动蛋白细胞骨架动力学的关键分子的活性。该发现可作为阐明BFA作用于细胞骨架的分子机制的基础。[1]

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adp核糖基化是一种翻译后修饰，可调节许多靶蛋白的功能。我们之前的研究表明，真菌毒素brefeldin A （BFA）诱导c -末端结合蛋白-1短形式/BFA- adp -核糖基化底物（CtBP1-S/BARS）的adp -核糖基化，CtBP1-S/BARS是一种双功能蛋白，在细胞核中作为转录因子，在细胞质中作为高尔基复合体胞内运输和有丝分裂分配过程中膜裂变的调节剂。在这里，我们报道了BFA对CtBP1-S/BARS的adp -核糖基化是通过一种非常规的机制发生的，该机制包括两个步骤：(i)由adp -核糖环化酶CD38合成BFA- adp -核糖缀合物；（ii） BFA- adp -核糖缀合物与CtBP1-S/BARS NAD（+）结合口袋的共价结合。这导致CtBP1-S/BARS锁定在二聚体构象中，从而阻止其与已知参与膜裂变的相互作用物结合，从而抑制有丝分裂高尔基分割中涉及的裂变机制。由于这种抑制可能导致G2细胞周期的停滞，这些发现为设计表达高水平CD38的肿瘤细胞细胞周期的药物阻滞剂提供了策略。［４］

Brefeldin A (BFA)是一种在真核细胞中诱导内质网（ER）应激的霉菌毒素。我们发现，与粘附培养相比，亚微克/毫升浓度的BFA在MDA-MB-231悬浮培养物（EC50:0.016µg/mL）中优先诱导细胞死亡。BFA还有效地抑制了MDA-MB-231细胞的克隆形成活性和迁移以及基质金属蛋白酶-9（MMP-9）活性。Western印迹分析表明，BFA的作用可能是通过下调乳腺CSC标志物CD44和抗凋亡蛋白Bcl-2和Mcl-1，以及逆转上皮间质转化来介导的。此外，BFA对悬浮的MDA-MB-468细胞也表现出选择性细胞毒性，并抑制了T47D和MDA-MB-453细胞中的瘤球形成，表明BFA可能对各种表型的乳腺癌症细胞有效[2]。

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2D克隆实验[2]
0-50 μg/mL 的Brefeldin A (BFA)预处理24 h后，以每孔1 × 103个细胞的密度在6孔板中重新接种，再培养12 d，每3 d换一次培养基。用甲醇-乙酸（3:1）固定菌落15 min，室温结晶紫（1%）染色30 min。

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创面愈合动力试验[2]
使用p10微移液管尖端在六孔板上进行过夜融合培养。细胞碎片用磷酸盐缓冲盐水（PBS）洗涤三次后，补充含有0-50 μg/mL Brefeldin A (BFA)的完整培养基。在伤口愈合后的指定时间，用相差显微镜捕捉伤口愈合的图像。

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明胶酶谱[2]
0-50 μg/mL Brefeldin A (BFA)处理细胞培养24 h，收集上清，0.22 μm过滤器过滤，Centricon自旋柱浓缩50倍，10 kD截止。用含有0.1% SDS和1mg /mL明胶的10%聚丙烯酰胺凝胶在非还原性SDS- page上分离浓缩上清。电泳后，用含有0.15 M NaCl、5 mM CaC12、5 μM ZnCl、0.02% NaN3、0.25% Triton X-100的50 mM Tris-HCl （pH 7.5）在室温下洗涤3次，每次洗涤30 min，然后在不含Triton X-100的同一缓冲液中37℃孵育20 h。采用考马西亮蓝R-250染色显示MMPs形成的明胶清晰区。

PK study [8]
Female SD rats (5 per group) were dosed intravenously with M-BFA BFA encapsulated in mixed nanomicelles based on TPGS and F127 copolymers) in 10% solutol HS-15% and 90% saline (v/v) at dose level of 520 mg/kg. Blood samples were collected from all of the animals at predose and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h postdose into tubes containing heparin sodium and 200 mM DDPV. Plasma was separated from the blood by centrifugation at 6800 rpm for 6 min at 4 °C and stored at − 80 °C until analysis. Method development and biological samples analysis were performed by Triple Quad 5500 LC-MS/MS with verapamil as an internal standard (Table S2). PK parameters derived from concentration–time profiles containing T1/2, Cmax, AUC(0−t), AUC(0-∞) were calculated using Phoenix WinNonlin 7.0 by the Study Director.

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Animal treatment and tumor inhibition in vivo [8]
Female BALB/c mice (20 ± 2 g, 5–6 weeks) randomly divided into three groups (5 per group). HepG2 cells (1 × 107/mouse) were implanted subcutaneously into the under back area of mice to establish HepG2 tumor model. The treated mice were checked daily to investigate the size changes of tumors after implanted the HepG2 cells. When the average tumor volume reached around 100 mm3 (volume = (tumor length) × (tumor width)2/2), all mice were ready for the subsequent studies. The HepG2 tumor-bearing nude mice were administrated with M-BFA every day for 14 days. PBS solution was used as the control group. The dosage of BFA in other groups was 5 mg/kg and 10 mg/kg body weight. The tumor size of each mouse was measured every 2 days. Tumor volume (V) was determined by the following equation: V = L × W2/2, where L and W are length and width of the tumor, respectively. The mice were anesthetized with diethyl ether at the end of experiment. The excised organs and tumor tissues were washed with cold PBS (pH 7.4) and were weighed and photographed.

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In vivo fluorescence imaging of tumor [8]
The biodistribution of M-BFA was observed by in vivo imaging. Amphiphilic ICG (Fig. S11) was dissolved in water (1 mg/mL), and then directly added in M-BFA solution (80 μg/mL). ICG molecules entered the nanomicelles via hydrophobic interaction. The mice were intravenously injected with 100 μL free ICG (100 μg/mL) and ICG-loaded M-BFA (containing 100 μg/mL ICG). The mice were anesthetized and imaged using a 808 nm excitation laser at predetermined time by the IVIS Spectrum image system. After 48 h, the mice were sacrificed and various organs were collected to image the fluorescence distribution.

In vivo pharmacokinetic study [8]
The initial characterization of Brefeldin A (BFA) plasma pharmacokinetics (PK) revealed that BFA decreased quite rapidly in an apparent biexponential manner in the mouse. The biological half-life (T1/2) of BFA in the body was 0.17 h [53]. On the basis of these, PK properties of M-BFA were evaluated in Sprague-Dawley (SD) rats. After intravenous administration dosing at 520 mg/kg (equivalent to BFA 20 mg/kg), the concentrations of M-BFA in plasma were analyzed. The results showed that M-BFA demonstrated moderate PK profile, the T1/2 was approximately 0.35 h (vs 0.17 h of BFA) and the maximum plasma concentration (Cmax) was 4065.68 ng/mL. It achieved a sufficient plasma exposure in rats, with an area under the concentration–time curve (AUC0−t) value of 3153.75 h*ng/mL.

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Biodistribution of M-BFA in tumor-bearing mice [8]
Given that Brefeldin A (BFA) does not have autofluorescence, to analyze the in vivo biodistribution and tumor-targeting efficacy of M-BFA, the indocyanine green (ICG)-loaded M-BFA was employed and injected to HepG2 tumor-bearing mice for optical imaging analysis. Fluorescence imaging was carried out at 0, 1, 2, 4, 8, 24 and 48 h after injection. As shown in Fig. 6A, after 8 h, ICG fluorescence was observed in tumor site in ICG-loaded M-BFA group. After 24 h, there was a sharp contrast between the accumulation of ICG-loaded M-BFA and free ICG in tumor tissue. The intensity of fluorescence increased to the strongest at this time point and remained strong after injected 48 h. In contrast, no obvious fluorescence was found in the tumor site in free ICG group. We further observed that the fluorescence mainly distributed in the liver in the initial stage. After injected 48 h, the fluorescence disappeared in the whole mice body.

mouse LD50 intraperitoneal 250 mg/kg Japanese Journal of Antibiotics., 34(51), 1981 [PMID:7241806]

[1]. Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons. Eur J Cell Biol. 1999 Jan;78(1):1-14.

[2]. Brefeldin A reduces anchorage-independent survival, cancer stem cell potential and migration of MDA-MB-231 human breast cancer cells. Molecules. 2014 Oct 29;19(11):17464-77.

[3]. Erythroleukemia cells acquire an alternative mitophagy capability. Sci Rep. 2016 Apr 19;6:24641.

[4]. Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS. Proc Natl Acad Sci U S A. 2013 Jun 11;110(24):9794-9.

[5]. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015 Feb 5;16(2):142-7.

[6]. Subcellular localization of herpes simplex virus type 1 UL51 protein and role of palmitoylation in Golgi apparatus targeting. J Virol. 2003 Mar;77(5):3204-16.

[7]. A time-related study of Brefeldin A effects in HSV-1 infected cultured human fibroblasts. APMIS. 1995;103(7-8):530-539. doi:10.1111/j.1699-0463.1995.tb01402.x.

[8]. Brefeldin A delivery nanomicelles in hepatocellular carcinoma therapy: Characterization, cytotoxic evaluation in vitro, and antitumor efficiency in vivo. Pharmacol Res . 2021 Oct:172:105800.

Brefeldin A is a metabolite from Penicillium brefeldianum that exhibits a wide range of antibiotic activity. It has a role as a Penicillium metabolite.
A metabolite from Penicillium brefeldianum that exhibits a wide range of antibiotic activity.
brefeldin A has been reported in Penicillium camemberti, Penicillium brefeldianum, and other organisms with data available.
A fungal metabolite which is a macrocyclic lactone exhibiting a wide range of antibiotic activity.

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Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor Brefeldin A (BFA) or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.[3]

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Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor Brefeldin A (BFA) or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.[6]

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Hepatocellular carcinoma (HCC) is one of the major cancers with high mortality rate. Traditional drugs used in clinic are usually limited by the drug resistance and side effect and novel agents are still needed. Macrolide brefeldin A (BFA) is a well-known lead compound in cancer chemotherapy, however, with poor solubility and instability. In this study, to overcome these disadvantages, BFA was encapsulated in mixed nanomicelles based on TPGS and F127 copolymers (M-BFA). M-BFA was conferred high solubility, colloidal stability, and capability of sustained release of intact BFA. In vitro, M-BFA markedly inhibited the proliferation, induced G0/G1 phase arrest, and caspase-dependent apoptosis in human liver carcinoma HepG2 cells. Moreover, M-BFA also induced autophagic cell death via Akt/mTOR and ERK pathways. In HepG2 tumor-bearing xenograft mice, indocyanine green (ICG) as a fluorescent probe loaded in M-BFA distributed to the tumor tissue rapidly, prolonged the blood circulation, and improved the tumor accumulation capacity. More importantly, M-BFA (10 mg/kg) dramatically delayed the tumor progression and induced extensive necrosis of the tumor tissues. Taken together, the present work suggests that M-BFA has promising potential in HCC therapy.[8]

C16H24O4

280.36

280.167

C, 68.55; H, 8.63; O, 22.83

20350-15-6

5287620

Typically exists as White to off-white solids at room temperature

1.1±0.1 g/cm3

492.7±45.0 °C at 760 mmHg

200-205ºC

180.8±22.2 °C

0.0±2.8 mmHg at 25°C

1.513

1.61

66.76

2

4

0

20

388

5

O([H])[C@]1([H])C([H])([H])[C@]2([H])C([H])=C([H])C([H])([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])OC(C([H])=C([H])C([H])([C@@]2([H])C1([H])[H])O[H])=O |c:10,t:31|

KQNZDYYTLMIZCT-KQPMLPITSA-N

InChI=1S/C16H24O4/c1-11-5-3-2-4-6-12-9-13(17)10-14(12)15(18)7-8-16(19)20-11/h4,6-8,11-15,17-18H,2-3,5,9-10H2,1H3/b6-4+,8-7+/t11-,12+,13-,14+,15+/m0/s1

(1S,2E,7S,10E,12R,13R,15S)-12,15-Dihydroxy-7-methyl-8-oxabicyclo[11.3.0]hexadeca-2,10-dien-9-one

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 (8.92 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 (8.92 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 (8.92 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (8.92 mM) (饱和度未知) in 10% EtOH + 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 EtOH 储备液加入400 μL PEG300 中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 5 中的溶解度: ≥ 2.5 mg/mL (8.92 mM) (饱和度未知) in 10% EtOH + 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100μL 25.0mg/mL澄清EtOH储备液加入到900μL 20％SBE-β-CD生理盐水中，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 6 中的溶解度: ≥ 2.5 mg/mL (8.92 mM) (饱和度未知) in 10% EtOH + 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 澄清乙醇储备液加入到 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。