SMCy5.5

Guide (The following is our recommended protocol. This protocol is provided as a guide only and should be modified according to your specific needs)^[1].
1. Cell Seeding: Seed cells in culture flasks one day before lipid droplet staining.
2. Pre-staining Treatment: Remove the culture medium the following day and wash adherent cells twice with PBS.
3. Dye Incubation: Incubate cells with SMCy3.5 or SMCy5.5 (prepared in OptiMEM containing 0.5% DMSO, 1 μM) at 37°C, 90% humidity, and 5% CO2 for 3 h.
4. Dye Removal: After incubation, discard the dye-containing culture medium, wash cells twice with PBS, add growth medium to ensure thorough removal of excess dye, and wash again with PBS.
5. Cell Processing: Digest cells with trypsin, centrifuge, resuspend cells in growth medium, and centrifuge and resuspend again.
6. Cell Mixing and Seeding: Mix the two cell suspensions thoroughly in equal volumes and seed them into IBiDi chambered coverglasses.
7. Nuclear Staining: Incubate cells with Hoechst (5 μg/mL) for 15 min for nuclear staining.
8. Fixation and Washing: Fix cells with 4% paraformaldehyde for 5 min, then wash three times with HBSS.
9. Imaging Time: Imaging is performed 18 h and 48 h after cell attachment.
Imaging Equipment and Parameters: A Nikon Ti-E inverted microscope was used; microscope settings are as follows: Hoechst (excitation 405 nm, emission 510 nm), SMCy3.5 (excitation 532 nm, emission 600 nm), SMCy5.5 (excitation 638 nm, emission 705 nm).
10. Image Recording and Processing: Images are recorded using NIS Elements software and then processed using Icy software.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.