1. Prepare Laurdan Stock Stain.

1.1. Prepare Laurdan Stock Stain Solution. (DMSO, concentration 1.8 mM. Reserve Solution ApplicationUnused solution should be aliquoted and frozen at a temperature of at least -20°C. It is important to avoid repeated freeze/thaw cycles. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)

1.2. Prior to use, the Laurdan Master Mix should be diluted at a ratio of 1:1000 or 1:200.

Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.

2. The following is a detailed description of the Laurdan staining process. NIH3T3 cells are cultured in a specialised medium, known as Duchenne's modified Eagle's medium, which is augmented with key ingredients such as 10% foetal bovine serum, 5 mL Pen-Strep, and 2.5 mL 1 M HEPES.

2.2. Freshly isolated NIH3T3 cells were inoculated into fibronectin-coated 35 mm Mattek glass-bottomed petri dishes.

2.3. The cells were then incubated with Laurdan at 37°C for 40 minutes.