BODIPY 493/503 中性脂滴荧光探针

产品标签

BODIPY 493/503；Nile Red 尼罗红；Neutral lipds 中性脂滴；DiI 细胞膜探针；DiR； CAS NO：121207-31-6

产品信息

产品描述

BODIPY 493/503是一种亲脂性荧光探针，定位在极性脂上，能用于标记细胞中性脂内容物，特别定位在活细胞和固定细胞的脂滴上。BODIPY 493/503与落射荧光显微镜、共聚焦荧光显微镜和双质子荧光显微镜，以及流式细胞仪兼容。最大激发和发射波长分别是493/503nm，适用于活细胞和固定细胞标记。

产品特性

1）CAS NO.：121207-31-6

2）化学名：(T-4)-[2-[1-(3,5-dimethyl-2H-pyrrol-2-ylidene-κN)ethyl]-3,5-dimethyl-1H-pyrrolato-κN]difluoro-boron

3）同义名：4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene

4）分子式：C₁₄H₁₇BF₂N₂

5）分子量：262.1

6）纯度：≥98%

7）Ex/Em：493/503nm

8）溶解性：溶于DMSO或无水乙醇

9）化学结构式：

保存与运输方法

保存：-20ºC避光干燥保存，至少2年有效。 

运输：冰袋运输。

储存液的制备和保存

1）将低温保存的10mg BODIPY 493/503（Mw:262.1）置于室温回温至少20min，低速离心后加入一定量的无水乙醇或无水DMSO配制成适量浓度的母液，比如5mM（往10mg 粉末加入7.63ml DMSO，充分溶解即可）。根据单次用量将储存液分装，≤-20℃避光干燥保存。需注意溶液内湿度的逐渐积累会随着时间引起探针聚集，从而务必干燥保存储存液。

2）如果想长期保存，可以用无水乙醇溶解粉末，之后分装到小量，之后使用真空泵来挥发掉乙醇。之后置于≤-20℃避光干燥保存。

探针的标记（仅作参考）

由于BODIPY 493/503属于疏水染料，难以快速的分散进入水溶性溶液中，为了能均匀稳定的标记细胞，可参考以下方法标记活细胞。

应用示例

图1. 胞内中性LDs的定量和观察。A，A498细胞在含BSA（0.2%）或含BSA的油酸过夜培养。之后根据流式分析的操作步骤进行BODIPY染色；B，A498细胞在含BSA（0.2%）或含BSA的油酸过夜培养。之后根据荧光分析的操作步骤进行BODIPY染色；

BODIPY staining for flow cytometry

1. Grow cells under culture conditions relevant for the study. A 35 mm dish/well is sufficient for the cell numbers required in this assay. For our assays, 50,000 A498 cells in 35 mm well were sufficient.

a. Overnight incubation of cells with 30 μM oleic acid can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control.

2. At the time-point of interest, prepare 2 μM BODIPY staining solution in PBS. The volume of staining solution required for each sample corresponds to the volume of media used for culturing cells.

3. Wash cells with a quick rinse using 3 ml PBS to remove media/serum.

4. Incubate on BODIPY staining solution in the dark for 15 min at 37 °C. Include an unstained control for flow cytometry.

Note: From this point, protect samples from light as much as possible.

5. Wash cells with a quick rinse using 3 ml PBS to remove staining solution.

6. Trypsinize cells to generate a single cell suspension. For the A498 cell line used in this protocol, cells were incubated with Trypsin-EDTA (0.25%) for 5 min at 37 °C.

7. Add 5 ml of PBS and transfer cell suspension to a 15 ml conical tube.

8. Pellet cells at 250 × g, 5 min, 4 °C.

9. Aspirate supernatant, wash the cell pellet with a quick rinse using 3 ml PBS, and pellet cells at 250 × g, 5 min, 4 °C.

10. Carefully aspirate the supernatant and resuspend cells in 300 μl 1× flow cytometry buffer.

11. Pass cell suspension through a 35 μm filter into a FACS tube.

12. Perform flow cytometry. Obtain a minimum of 10,000 events per condition.

13. The investigator can analyze data as mean fluorescence (Figure A) or display the data as a histogram (Figure B).

注意事项

1）荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。

2）为了您的安全和健康，请穿实验服并戴一次性手套操作。

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上海懋康生物科技有限公司是一家涉足于生命科学和生物技术领域研究的试剂、仪器和实验室消耗品与实验服务工作，主要从事细胞生物学、植物学、分子生物学、免疫学、生物化学、蛋白组学。生物制药与诊断试剂研发生产等领域。 本公司秉承“以人为本，以诚为信、合同守信”的经营理念。坚持"品质保障"的原则为广大客户提供优质产品。

引用文献：

[1]Jinting Liu, Yihong Wei, Wenbo Jia, Can Can, Ruiqing Wang, Xinyu Yang, Chaoyang Gu, Fabao Liu, Chunyan Ji, Daoxin Ma, Chenodeoxycholic acid suppresses AML progression through promoting lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway and inhibiting M2 macrophage polarization, Redox Biology, Volume 56,

2022,102452,ISSN 2213-2317,https://doi.org/10.1016/j.redox.2022.102452.

BODIPY 493/503 analysis

We first treated THP1 and Molm-13 cells with 0, 0.12 and 0.17 mmol/L CDCA for 24h. Then AML cells were gathered and incubated with 1 μmol/L BODIPY 493/503 (Shanghai Maokang Biotechnology, China) for 30 min at 37 °C. After being washed twice by PBS, the cells were counterstained with DAPI and covered with glass slides for confocal microscope detection. The mean fluorescence intensity was used for analysis. Besides, AML cells were pretreated with A922500 for 1h before CDCA treatment to examine the level of LDs using flow cytometry.

[2] Qin T, Liu Z, Wang J, Xia J, Liu S, Jia Y, Liu H, Li K. Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling. Cancer Biol Med. 2020 Aug 15;17(3):753-767. doi: 10.20892/j.issn.2095-3941.2020.0024. PMID: 32944404; PMCID: PMC7476093.

Neutral lipid droplet fluorescence staining

BODIPY 493/503 (Shanghai Maokang Biotechnology, Shanghai, China) is a commonly-used agent for staining intracellular lipid droplets to monitor lipid storage (15,16). Briefly, the cells were seeded on coverslips and stained with BODIPY 493/503 at 2 µM final concentration for 15 min at 37 ℃ in the dark according to the manufacturer’s instructions. Cells were then briefly washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), stained with 4’,6-diamidino-2-phenylindole (DAPI; Dojindo, Gaithersburg, MD, USA; D523) for 5 min at room temperature in the dark. The images were captured and analyzed by an Olympus FluoView 10i confocal microscope (Olympus, Tokyo, Japan).