Throughout the past few weeks, I have been in contact with both ATCC and NanoEnTek regarding the NBT-II cell line. After further analyzation of the protocol provided to me by ATCC technical support and cross-comparison with my own procedures carried out, it was clear that two steps were not carried out. The first instance was not washing the cell lines in Dulbecco Phosphate Buffered Saline. DPBS removes excess serum left behind from medium and inhibits or lengthen the trypsinization period possibly preventing the adherent cells from completely detaching from the bottom of the T-75 flasks. Additionally, during the initial subculturing carried out I was supposed to centrifuge the complete growth medium from the T-75 flask at 125xg for 5-7 minutes and then resuspend these cells in fresh media as an additional culture to keep going. Additionally, I will be purchasing more of the Eve NanoEnTek cell counting slides to further test the viability of my new cells following my initial culturing.
I was hoping to reculture the cells last week or this week, however after working tirelessly to find someone to carry out the subculturing procedures on a regular basis during the 3 week period that I will be gone. It became evident that repurchasing these cells at this time would be futile. As a result, I will be purchasing these cells alongside the DPBS as soon as I return from break to finally carry out research. This process has been extremely exhausting and over the course of spring break, I plan on revising and reviewing the subculturing techniques alongside the overall plan for my research with my mentor (Dr. Pal).