meso-Benzothiazole-BODIPY 505/515

Fluorescent probe/dye

细胞标记实验方案（指南）
注：本方案仅作为推荐指南，应根据具体实验需求进行调整[1]。
操作步骤：
1. 细胞准备
○ 以5×10⁴个细胞/mL的密度将细胞接种于20mm共聚焦培养皿中。
○ 按标准培养条件孵育细胞。
2. 探针标记
○ 用于共聚焦成像时，在培养基中加入5μM的meso-苯并噻唑-BODIPY 505/515（探针1）。
○ 用探针孵育细胞30分钟。
3. 成像参数
○ 激发波长：488nm
○ 发射光收集范围：520-600nm

免疫荧光[1]
细胞类型： SH-SY5Y 细胞
测试浓度： 5 μM
孵育时间： 30分钟
实验结果：在低粘度细胞中表现出相对较弱的荧光发射，但当SH-SY5Y细胞与LPS和制霉菌素预孵育时表现出较强的荧光发射。

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细胞粘度成像和自噬监测[1]
共聚焦成像使用20 mm共聚焦皿进行，SH-SY5Y细胞为5×104个细胞/mL。对于亚细胞成像，使用Dulbecco改良的Eagle培养基（DMEM）中的1（5μM）和Mito tracker Blue或Lyso tracker Blue（1μM）培养细胞30分钟。用磷酸缓冲盐水（PBS）洗涤细胞三次后，使用Leica TCS SP8共聚焦显微镜用100倍油浸物镜进行亚细胞成像。对于Mito tracker Blue或Lyso tracker Blue，细胞在405 nm处被激发，在425-500 nm范围内收集发射，该范围被标记为蓝色通道。对于1，细胞在488 nm处被激发，在520-600 nm处收集发射，这被标记为红色通道。合并后的蓝色和红色通道也已给出。

对于粘度响应共聚焦成像，细胞首先与无、脂多糖（LPS）或制霉菌素（均为20μM）一起培养40分钟。用PBS洗涤三次后，将新鲜制备的1（DMEM中的5μM）储备溶液加入上述细胞板中，在37°C下进一步培养30分钟。细胞在488nm下激发，用63倍油浸物镜在520-600nm下收集发射物。

为了监测活细胞中1的粘度变化，使用了低温和地塞米松。细胞首先分别在37和4°C下用1（10μM）处理30分钟。另外两组首先在37°C下用1（10μM）孵育30分钟，然后用地塞米松（100μM）或二甲亚砜（DMSO，10μL）再处理10分钟。用PBS洗涤三次后，细胞在488 nm处激发，用100倍油浸物镜在520-600 nm处收集发射。

为了通过溶酶体粘度变化监测自噬过程，将SH-SY5Y细胞与1（10μM）在37°C下孵育30分钟，然后在Hank's平衡盐溶液（HBSS）、正常培养基或添加了3-甲基腺嘌呤（3-MA）（一种自噬抑制剂）的HBSS中培养2小时，分别给予饥饿条件、丰富营养条件或自噬抑制条件。细胞在488nm处被激发，用100倍油浸物镜在520-600nm处收集发射。

[1]. Novel Meso-Benzothiazole-Substituted BODIPY-Based AIE Fluorescent Rotor for Imaging Lysosomal Viscosity and Monitoring Autophagy. Anal Chem. 2022 Oct 25;94(42):14707-14715.

Meta-substituted boron dipyrrole methylene (BODIPY) offers a potential and innovative strategy for the synergistic construction of aggregation-induced emission (AIE) probes and fluorescent rotors for monitoring changes in cell viscosity, which is crucial for understanding the role of viscosity in closely related diseases. Therefore, we have for the first time rationally designed and synthesized a BODIPY-based fluorescent probe (1) with a rotatable meta-benzothiazole group, exhibiting good viscosity responsiveness and AIE properties. Probe 1, through direct attachment to the thiazole group, shows almost no emission in low-viscosity solvents; however, a strong emission peak appears at 534 nm, gradually increasing with increasing viscosity, attributed to the effective confinement of the rotatable meta-benzothiazole group. In methanol/glycerol mixtures, with viscosities ranging from 0.59 to 945 cP, the fluorescence intensity (log I534) exhibits a good linear relationship with viscosity (log η). Interestingly, compound 1 shows a higher emission intensity at 534 nm in a 70% aqueous solution than in pure acetonitrile solution, likely due to aggregation-induced rotational inhibition. Cell imaging showed that compound 1 was able to successfully sense changes in lysosomal viscosity induced by lipopolysaccharide, nystatin, low temperature and dexamethasone in living cells, which could be further applied to monitor autophagy by tracking changes in viscosity. In contrast, its analog 2, which is directly linked to a phenyl group, did not show viscosity response or aggregation-induced emission (AIE) properties. Therefore, we report for the first time a fluorescent rotor based on mesobenzothiazole-BODIPY that has aggregation-induced emission (AIE) and lysosomal viscosity response properties in nerve cells and can be further applied to autophagy monitoring. This work provides an innovative strategy for designing potential AIE and viscosity-responsive probes. [1]

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In summary, we introduce a rotatable lysosomal-targeting benzothiazole group into the BODIPY core for the first time, and report an AIE fluorescent probe based on mesobenzothiazole BODIPY that can use the free rotation of the mesobenzothiazole group to image lysosomal viscosity in living cells and monitor autophagy. By directly attaching the mesobenzothiazole group to the thiazole moiety, probe 1 exhibited a redshift in both absorption and emission spectra compared to probe 2, which was directly attached via a phenyl group. More interestingly, even with slightly different attachment methods, probe 1 and probe 2 displayed distinctly different properties, such as viscosity responsiveness and aggregation-induced emission (AIE) characteristics. At low viscosity, probe 1 showed almost no fluorescence emission; as viscosity gradually increased, a strong fluorescence emission band appeared at 534 nm. Notably, this meta-benzothiazole-substituted probe exhibited excellent AIE characteristics at 534 nm, further confirming that the rotation of the meta-benzothiazole group can be effectively restricted in both aggregated states and high viscosity environments. Further cell experiments showed that the meta-benzothiazole group can also serve as a lysosomal targeting group, and its application in cell viscosity monitoring was successful after pretreatment with LPS, nystatin, low temperature, and dexamethasone. Furthermore, the viscosity-responsive properties of probe 1 were also applied to autophagy monitoring. In summary, we first used the uncommon meta-phenyl substitution to introduce the meta-benzothiazole group into the core of BODIPY by direct connection with a five-membered ring, studied its viscosity/aggregation-induced emission (AIE) properties, and further applied it to the visualization of cell viscosity changes and the monitoring of lysosomal autophagy. Other related studies on BODIPY based on meta-five-membered heterocyclic substitution are underway, aiming to optimize its AIE/viscosity properties and study its structure-property relationship. [1]

C20H18BF2N3S

381.25

Typically exists as solid at room temperature

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。

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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。

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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)

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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。

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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

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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。

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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。