C12 NBD Sphingomyelin

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs)^[2].
1. Preparation of C12 NBD-Sphingomyelin-BSA Complex (for Cell Labeling)
1.1 Dissolve lipids: Prepare a 1 mM C12 NBD-Sphingomyelin stock solution using anhydrous ethanol.
1.2 Dried Lipids: Add 50 μL of the 1 mM stock solution into a glass tube, then blow dry it with nitrogen, and subsequently perform vacuum drying for 1 hour.
1.3 Formation of complex with BSA: Reconstitute the dried lipid with 200 μL anhydrous ethanol; take 10 mL of HBSS/HEPES solution containing 0.34 mg/mL defatted BSA, and slowly add the above ethanol solution of NBD-Sphingomyelin while vigorously vortexing.
2. Cell Staining Procedure
2.1 Cell Washing: Use HBSS/HEPES buffer to wash the cells that are growing on the cover glass and have a confluence of 70-80%.
2.2 Incubation: Add a staining solution of NBD-Sphingomyelin/BSA complex at a concentration of 5 μM to the cells.
2.3 Temperature Control: Incubate at 4℃ for 30 minutes to allow the probe to penetrate the cell membrane while minimizing endocytosis within the cells.
2.4 Washing: Remove the culture medium and wash the cells multiple times with ice-cold culture medium to remove the unbound probes.
2.5 Imaging and Analysis: Observations are conducted using a fluorescence microscope (excitation wavelength Ex = 470 nm, emission wavelength Em = 525 nm); if uptake studies are to be carried out, the cells can be maintained at 37°C for further processing.

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