To begin, obtain 80%grown bovine hepatocyte cells and replace the culture medium with DMEM containing linoleic acid. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for 24 hours to accumulate the LDs. Then remove the culture medium and wash the adherence cells with PBS.
Add BODIPY neutral fluorescent probe in the cells and incubate in the dark for 30 minutes. After three PBS washes, add DMEM containing linoleic acid to the cells. Place the culture dish in the groove of the microscope of the living cell station and turn on the microscope and computer.
Run NIS-Elements software. Find the field of view at 4x magnification. Then adjust it to the 40x, turn PFS on, refocus, and select the appropriate field of view.
For the test run, set the appropriate time and channels. Then press Run Now to shoot the samples for one minute. For the experiment, in the Time tab, set the interval time of five minutes and shoot duration of six hours, set fluorescent and bright-field channels, and press Run now to shoot the samples.
Then choose File followed by Save As and AVI Image File format to export the data to a video in AVI format. Large LDs were formed through the fusion of small LDs. In the first 15 minutes, there were smaller LDs.
From 20 minutes, they started to fuse and were completely fused into larger LDS by 35 minutes.
