Take #3

For background info read:

Second is the best...

Science of mistakes and caveole

Everytime I run an assay, there are more things that still end up going wrong. I know I shouldn't be mad about it, but the last experiment, the procedure and everything was right:I just got interesting results.

In this particular experiment, I wanted to measure cell death over time while the human dermal fibroblasts were submerged in hypoosmotic HBSS+HEPES buffer, just HBSS+HEPES buffer, in hydrogen peroxide, and in hypoosmotic buffer and hydrogen peroxide. In the previous experiment, there wasn't significant cell death, so I wanted to see when most cells would die. There was a 30 minute, a one hour, a two hour and a four hour interval.

I also switched out they typical MEM media for a HBSS and HEPES buffer because they help maintain the pH better at room conditions.

Basically, for this experiment I had three independent variables, time, condition and cell type. I was still comparing HDF and CMDF cells, but also how they had different behavior in different conditions over time. I had one condition where the cells were in a plain buffer (HBSS and HEPES), one where the cells were in hypoosmotic buffer and hydrogen peroxide, another group in plain hypoosmotic buffer and the last group in hydrogen peroxide; there was one experiment condition and three control conditions.

For this experiment, cells were analyzed by staining them with Trypan Blue and then using a cell counter to determine cell viability.

Like always, there was the unexpected, and the delays. Nothing terrible went wrong, it was just that the cell counter took longer than expected. In order to collect more precise results, I tested three separate samples from the same treatment three times each. Basically, I had to check my results nine times for each well. This took a long time and I was in back log. I started the experiment at around 10:00 am or so and didn't finish my experiment until 6:30 pm that Friday night. My arms were throbbing and stomach was growling until they went numb. I learned about the importance of designing a well balanced experiment that evening. There was no time to eat lunch, run to the bathroom or for a sip of water, it was continuous pipetting.

The results were puzzling. There were cells that underwent four hours of treatment that had the same, if not less cell death than the cells in the same condition for less time. This could've been caused by a variety of reasons: perhaps the cells were never dying, but simply had holes in their membranes that later fixed themselves, after letting the dye in. This is an interesting find because it could mean that the experiment isn't working.

Next steps:

1. Check to see if apoptosis and necrosis can be differentiated by using dyes that are specifically tagged for checking those functions.