Hello Marine Ma

BODIPY 493/503 is prone to photobleaching. The faster you capture the image, the more signal you will retain.

You may follow the below protocol.

1. Dilute the BODIPY 493/503 stock solution in sterile PBS to a final working concentration of 2µM.

2. Wash the HepG2 cells with PBS to remove culture medium and serum.

3. Add the 2µM BODIPY 493/503 solution to the cells and incubate for 15–30 minutes at 37°C in the dark.

4. After incubation, wash the cells once with PBS to remove excess dye and visualize the cells immediately under the microscope to capture the bright signal before it photobleaches.

5. Use a green channel filter (excitation around 494 nm) to excite the dye and capture its emission.

Precautions to be taken.

a. Protect the stained cells from light after incubation to prevent photobleaching.

b. Do not use methanol-based fixatives or detergents after BODIPY staining in order to preserve the dye signal and prevent its loss from lipid droplets, since methanol can extract phospholipids and compromise the dye's association with the lipid droplets. Alternatively, for a more robust protocol, you may use an aldehyde-based fixative such as paraformaldehyde to preserve cell and tissue structures. Aldehydes cross-link proteins, stabilizing cellular components without extracting lipids or damaging the BODIPY signal.

Best,

The MCE laboratory has tested the quenching rate of this dye. We used BODIPY 493/503 to stain HepG2 cells, and fluorescence detection results showed that under the green fluorescence channel, the fluorescence intensity was relatively strong right when the laser was first applied. After about 10 seconds, the fluorescence intensity slightly decreased, but clear lipid droplet staining could still be observed after 5 minutes.

Here are some suggestions for you: if rapid quenching is observed during imaging, you can reduce the laser intensity, shorten the exposure time, and minimize laser exposure when not actively capturing images.

For example, under the eyepiece, first adjust the focus using brightfield, then switch to the channel corresponding to the dye. Locate the field of view under low-intensity illumination, then switch to capturing fluorescence images on the computer. Based on the results, fine-tune parameters and capture images in different fields of view.

The specific procedure is as follows:

• Stock solution: 10 mM in DMSO;
• Working solution: 10 μM in serum-free DMEM medium;
• Staining: stain for 30 minutes; stain with Hoechst 33258 (HY-15558) for 3–10 minutes. Protect from light.

The answer to this question is provided by MedChemExpress Technical Support. [email protected]