March 27-31
This week, I spent the majority of my time optimizing Promag iron labelling with the 231 BR cell line. I cultured three T75 flasks each containing 200,000 231 BR cells and labelled each flask with 25 ug/mL, 50 ug/mL, and 75 ug/mL of Promag, respectively. I learned a lot this week by running this experiment. First, I learned how to calculate the loading concentration of iron for cell labelling.
The calculation is:
loading concentration = [concentration of iron (provided by manufacturer) x the volume of iron added to the cell culture] / the total volume of media in the cell culture flask
For my experiment, I was given three loading concentrations to try (25, 50, and 75 ug/mL of iron). I was also given the iron concentration of Promag (4.79 ug/uL). I reworked the equation to find the volume of iron to add to the cell culture for each respective loading concentration. Then, I added the calculated volume of Promag to 10 mL of media and added this mixture to each cell culture flask. The 231 BR cells were incubated overnight with the iron.
The next day, I counted the cells and used magnetic separation to get rid of unlabeled cells and debris. This step helps to optimize the cell labeling by separating out the labeled cell population from the unlabeled population. The next step was to take the labeled cell population and perform Pearls Prussian Blue (PPB) staining to check my labeling efficiency. I also saved 500,000 cells from each of the three different loading concentration groups for MPI. See below for some challenges that I faced during the PPB step, and how I overcame them.
Some challenges and reflections:
All of the BLI trials using the 231 BR cells have been unsuccessful at producing bioluminescence, even with very high cell numbers included (5x10^5, 1x10^6, and 2x10^6, respectively). These cells were checked on the fluorescent microscope for reporter gene (GFP) fluorescence, and there was no observed GFP fluorescence. This has led to the conclusion that the 231 BR cells were either not transfected well, or that the transfected population has disappeared. The existing cells are not luciferase positive. Upon reflection, it might be beneficial to run another Promag labelling trial with the specific luciferase positive cell line that will be used for the BLI and MPI experiment. I have recently been growing 4T1 luciferase positive cells containing GFP as a reporter gene. These cells have shown reporter gene fluorescence and success with BLI at cell numbers as low as 50,000 cells/well.
During the third day of my cell labelling experiment with the 231 BR cells, I miscalculated the number of cells that I needed to add to each cytospin funnel to be added to glass slides for PPB staining. Instead of 200,000 cells/slide, I accidentally loaded 20,000 cells/slide. Reflecting on this, I could have been more vigilant in checking over my calculations before loading the cells in the cytospin. This is something that I will keep in mind to improve on in the future to prevent having to repeat experiments. On Monday, I plan to stain slides that I prepped this Friday containing 200,000 cells/slide from each of the three loading concentration groups. I will image these slides to see which loading concentration was best for labelling with the 231 BR line.
That's all for now! I'm looking forward to learning MPI over the next few weeks :)