BODIPY 493/503 (Pyrromethene 546) is a fluorescentneutral lipid dye(Ex/Em: 493/503 nm). BODIPY 493/503 can facilitate quantification of neutral lipid content by flow cytometry and observation of lipid droplets (LDs) by microscopy^[1].

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
A. BODIY staining for flow cytometry
1. Grow cells under culture conditions relevant for the study.
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℃. 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.
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 ℃.
9. Aspirate supernatant, wash the cell pellet with a quick rinse using 3 mL PBS, and pellet cells at 250×g, 5 min, 4℃.
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 or display the data as a histogram^[1].
B. BODIY staining for microscopy
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℃ 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.
9. Incubate under the culture conditions relevant to your experiment.
10. At the time-point of interest, prepare 2 μM BODIPY staining solution in PBS.
11. Wash cells with 3 mL PBS.
12. Incubate on 3 mL staining solution for 15 min at 37 ℃. (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.
18. Allow the mounting solution to cure overnight at room temperature.
19. Slides can be stored at 4℃ or imaged immediately^[1].

262.11

Solid

C₁₄H₁₇BF₂N₂

121207-31-6

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

DMSO : ≥ 5 mg/mL(19.08 mM)

*"≥" means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80℃, 6 months; -20℃, 1 month (sealed storage, away from moisture)。-80℃ 储存时，请在 6 个月内使用，-20℃ 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照In Vitro方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    90%corn oil

  Solubility: ≥ 0.5 mg/mL (1.91 mM); Clear solution

  此方案可获得 ≥ 0.5 mg/mL (1.91 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 5.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。