March 20th - 24th
This week, I had the opportunity to practice BLI with a population of 231 BR cells that I have been culturing. These cells are Fluc positive and also express tdT and GFP reporter genes. I was instructed to load a 24-well plate with triplicates of the following cell amounts: 5,000 cells/well, 10,000 cells/well, 20,000 cells/well, and 500,000 cells/well.
While learning the BLI system in more depth this week, I learned some important optimization/image enhancement tips:
Triplicates containing the same cells/well should be plated in the same row, for clearer comparison, especially if the experiment will be added to a paper figure
A black well-plate should be used for paper figures because it is more clear when imaging with BLI, compared to the clear well plates
ROI tools can be used to enhance the image (such as binning and smoothing)
The analysis tool Prism graphs average radiance vs cell number for you, and it would be helpful to have access to this software when I am analyzing BLI data
One challenge that I faced this week with BLI was that the 231 BR cell line showed no observable signal after multiple trials. Initially, I thought that it was human error on my part, however this happened more than once after I had ensured that all steps were performed correctly. After some thinking and reflecting, the cell line was checked on the fluorescent scope for reporter gene fluorescence, which was not present. This means that the luciferase positive 231 BR population is not present. It was either not sorted properly, not transfected with luciferase properly, or it died out after multiple passages.
In the future, I will ensure to check luciferase positive cells on the fluorescent microscope for reporter gene fluorescence. This is a good step to perform with any cell line before BLI to ensure that luciferase is present within the given cell population. I will now start culturing a new cell line that is luciferase positive.