Second Is the Best (or so the annoying child rhyme says)

After the disaster with the first run, I decided to talk about the experiment protocol with my PI. We made some pretty big changes that simplified the experiment. Over winter break (12/21/17), I prepped all my materials and ran the second experiment. Unlike the first protocol, this time, I would use the Keyence Microscope to take pictures of the cells and manually count them. Also, I would be running the experiment in doubles instead of quadruplicates. Although there were some steps that occurred a few minutes later, this run was deffinatly less hectic than the last. And I did get some promising results (which I can't disclose). After the experiment, Jon, my new mentor gave me a few tweaks I could make to run a much reflective assay.

Here are some problems that occurred and the solutions to fix them:

1. During imaging after the treatment, the addition of Trypan blue, and the rinse with PBS, many dead, floating cells were observed. This could be due to the PBS rinse done to halt the effects of the treatment. Also, this leads to the question of dead cells being washed away when the treatment solution is aspirated.

2. Another confounding variable is the increase in pH due to the lack of CO2. In order for MEM media to keep the pH at the same level, it needs CO2. Since most of this experiment was done outside the incubator, the possible increase in pH is another confounding variable.

3. Another error found during this experiment was that the microscope took a longer time than expected. Since all the wells were on the same plate, there were a few steps that were late. For example the cells undergoing oxidative and hypo-osmotic stress spend 13 more minutes than planned in the H2O2 solution. In addition, due to the different endings for treatments, some cells were rinsed, underwent Trypan blue treatment, and kept in PBS solution for longer to avoid delays in the addition or removal of solution for other wells.

Solutions/moving forward:

1. A cell counter will be used to account for all the cells, including those alive and dead

2. Cells will be seeded in and the experiment will be done in HBSS + HEPES media

3. Experiment will be redesigned so that all cells reach the “counting step” at the same time. This way all wells don’t receive delayed treatments due to the time spent imaging another well. In addition, a hypo-osmotic time study will be done to find the ideal time at which there is significant cell death, to see if the time cells undergo hypo-osomtic stress could be increased, so that there is more time between steps, which will decrease the probability of wells receiving late treatment.