To begin, equilibrate the total triglyceride kit at room temperature for 20 minutes. Collect the supernatant of overnight grown Hep G2 cells and centrifuge at 1, 570 G for 10 minutes. Set the standard and test sample wells.
Add 50 microliters of the oleic acid to the standard labeled wells. In addition to the blank and standard wells, add 10 microliters of cell culture to the sample wells. Then add 40 microliters of sample diluent to each well.
Now add 100 microliters of detection antibody, horse radish peroxidase to each well. Seal with a plate membrane and incubate at 37 degrees Celsius in a constant temperature oven. After one hour, discard the supernatant and blot dry on dust-free paper.
Add washing solution to each well and incubate at room temperature for one minute. Now, add 50 microliters of substrate A and 50 microliters of substrate B to each well. Mix gently and incubate for 15 minutes at 37 degrees Celsius.
Add 50 microliters of termination solution, and within 15 minutes, measure the optical density at 450 nanometers. Plot the concentration of the standard along the x axis and the corresponding absorbance value along the Y axis. Perform linear regression and derive the curve equation to calculate the concentration value of each sample.
Compared to the control group, treatment of Hep G2 cells with varying concentrations of oleic acid resulted in a significant increase in the total triglycerides content of the cell supernatant.
