BODIPY 493/503染色实验步骤

基本信息：

BODIPY 493/503是一种亲脂性荧光探针，定位于极性脂质，可用于标记细胞中性脂质含量，特别是定位于脂滴的脂质含量。[1]，[2]BODIPY 493/503与落射荧光，共聚焦和双光子显微镜以及流式细胞仪兼容。它分别显示493/503 nm的激发/发射最大值，可用于活细胞和固定细胞应用。

货号：ajci70160
CAS：121207-31-6
分子式：C14H17BF2N2
分子量：262.1
纯度：＞99%

存储：Store at -20°C

溶解：可溶于DMSO（≤1mg/mL），可溶于DMF，可溶于氯仿，可溶于甲醇

BODIPY染色步骤用于流式细胞术：[2]

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.

  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 1A) or display the data as a histogram (Figure  1B).

BODIPY染色步骤用显微镜检测法：[2]

1.Autoclave coverslips in a glass bottle.

2.In the tissue culture hood, place coverslips into 35 mm cell culture dishes.

3.Prepare 2 mg/ml collagen solution in PBS.

4.Treat the coverslips with collagen to promote cell adherence. Add 3 ml collagen solution to culture dishes and incubate at 37 °C for 30 min.

Note: Use forceps to ensure that coverslips are flush with the bottom of the culture dish, eliminating any air bubbles that may be under the cover slips.

5.Aspirate the collagen solution.

6.Wash with PBS.

7.Add PBS to culture dishes and place under UV light in the culture hood to sterilize.

8.Plate cells into culture dishes containing the coverslips. The optimal cell number should be determined to achieve confluence of 30–50% at the time of staining to permit proper imaging. For A498 cells used in this protocol, 100,000 cells were plated in 35 mm wells to permit staining at 48 h post plating.

9.Incubate under the culture conditions relevant to your experiment.

   For this protocol, A498 cells were incubated in DMEM (high glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS at 37 °C.

  Overnight incubation of cells with 30 μM oleic acid with BSA 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.

10.At the time-point of interest, prepare 2 μM BODIPY staining solution in PBS.

  For this protocol, A498 cells were stained 48 h after plating, after an overnight incubation with BSA or BSA + oleic acid.

11.Wash cells with 3 ml PBS.

12.Incubate on 3 ml staining solution for 15 min at 37 °C.

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

13.Wash twice in 3 ml PBS.

14.Fix cells in 3 ml 4% PFA for 30 min at room temperature.

15.Remove 4% PFA.

16.Wash samples 3 × 5 min in PBS.

17.Use forceps to mount cover slips onto glass slides.

     Add a drop of Prolong® Gold antifade reagent with DAPI onto slide.

     Use forceps to pick up cover slips and place onto the drop of mounting solution, ensuring that the side that side with cells is placed face down onto the mounting solution.

18.Allow the mounting solution to cure overnight at room temperature.

19.Slides can be stored at 4 °C or imaged immediately (Figure 1C).

参考文献：

[1]. Listenberger, L.L., and Brown, D.G. Fluorescent Detection of Lipid Droplets and Associated Proteins. Curr. Protoc. Cell. Biol. Supplement 35, 24.22.21-24.22.11 (2007).

[2]. Qiu, B., and Simon, M.C. BODIPY 493/503 staining of neutral lipid droplets for microscopy and quantification by flow cytometry. Bio. Protoc. 6(17), (2016).

恭喜以下科学家使用我司BODIPY 493/503发表SCI文章：

1.Cell Press.cell host & microbe.16 October 2023（IF:30.3，Q1）.

2.Animals 2022, 12, 2616.

3.International Journal of Biological Macromolecules.Volume 248, 1 September 2023, 126025.

4.Cellular Signalling.Volume 111, November 2023, 110878.

我司bodipy493/503的质检谱图如下：

1.核磁氢谱，从检测HNMR可以看出产品无杂质峰，结构正确，纯度应＞99%

2.核磁碳谱，从检测CNMR可以看出碳的种类及含量与结构相符。

3.核磁氟谱，从检测FNMR可以看出仅仅只有一种氟结构，与结构相符。