I began the process of my research by culturing the complete growth medium for the initial culturing of the cells and future subculturing of the cells. Additionally, the cell line, NBT-ii, has come from ATCC and I have cultured it under the guidance of Teacher Leslie.
Tuesday is the first day of subculturing of the new cell line. Here, I will carry out a 1:2 split into T-75 flasks with a total volume of 15mL in each flask. Throughout the initial cultivation, I placed the vile of the NBT-ii cells into a 37 degree Celsius water bath to thaw out, then we used a 1:15 ratio where we put 1 mL of the NBT-ii contents in combination with 14 mL of the Eagle’s Minimum Essential Medium with 10% FBS. After the 4 days period in the incubator, in order to allow the cells to reach 70% confluency, I planned to sub cultivate the cells. Later, I plan on conducting my first test which will consist of pomegranate juice and control – this is because the other products (curcumin and grape seed extract) have yet to come. During my first subcultivation, I extracted the complete growth medium, and trypsinized the cells under a sterile hood and using sterile technique. Once trypsinized I placed the flask under the inverted microscope. I initially could not find anything, however, thanks to the help of Teacher Mariska she showed that the microscope was focused on the bottom on the cells rather than the trypsinized liquid (where we could see the cells). However, during this process, the cells were in trypsin for over 15 minutes which will most likely affect the viability of these cells. This will result in a longer period for the cells to adhere and possibly regain confluency. The cells are currently in the CO₂ incubator and will be continually checked each day under the inverted microscope to ensure that the cells are regaining viability.