The Science of Caves and Mistakes

Heart racing, hands sweating, glasses sliding off, I looked down at my two twenty four well plates, the conical tubes up ahead, tiny eppendorf tubes to the left and pipette tips to the right. I went through my mental list once again, thinking about the materials needed for every step. This was my first time running the complete version of my protocol and with my mentor leaving later that week and my internship on the line, there was no room for error. After taking a deep breath, I started the experiment.

Any other day, I might've talked about how long and boring an hour is, but in this experiment, an hour was not long at all. Every five minutes there were multiple steps to be completed, and random "mistakes" kept track of. For example there were some solutions collected into the wrong tubes. I had 40 wells with five different treatment plans. Three of those treatments were controls my other two treatments were the things I was probing for. The goal of the experiment was to see if there were any differences in resistance to osmotic pressure between dermal fibroblasts with and without a Caveolin-1 mutation (F160X). Caveolin - 1 is protein vital for the formation of caveolae or "little caves" in the plasma membrane. These structures allow the cell to expand as a response to hypo-osmotic stress.

My lab is researching caveolin and a caveolin mutation in a young girl with progeria, lipodystrophy and pulmonary hypertension. We believe that her early caveolin stop mutation, a mutation where her caveolin protein is twenty amino acids shorter than normal caveolin, decreases the mechanical protection offered by caveolae. In addition, reactive oxygen species such as hydrogen peroxide react with caveolin and inhibit the protein. The purpose of my experiment is to assess the difference in cell death due to hypo-osmotic and oxidative stress in dermal fibroblasts with and without the caveolin mutation.

At first this experiment seemed simple enough for a rookie, but after many discussions with various people, many control treatments were added. This experiment went from a simple easy task to one that required the best hand eye coordination and concentration for an hour. Since I was a rookie and this experiment had many steps every five minutes, it was vital that I had everything planned out prior to starting. I thought writing a materials list and labeling every mini eppendorff tube I would ever need would prevent any mistakes from occurring, but boy was I wrong.

After finishing the treatments, all the collected liquids and the cells were spun down using a centrifuge. In an ideal world, this procedure would cause cells to form a pellet at the bottom of the tube without killing any cells. In a hurry to go the bathroom and with a mind preoccupied with my hunger cramps (it was three and I didn't have a lunch yet and had a light breakfast), I failed to notice the tiny corner that read RPF. My heart beat speed and for a second the cold, dry lab became hot and humid. I could feel my cheeks getting red and tears stirring in my eyes. I was glad, for the first time that day that no one was there watching me. This experiment took months of preparation and I was afraid that all those people were right, I was too young to run my own experiment. Praying from the bottom of my heart, I decided to search up what RPF meant and if it was the same thing as Rotations per minute or RPM. I was hoping that the two terms were interchangble and that I didn't make a mistake. Normally, I would ask about every little detail, but who ever thought a centrifuge had so many options. I've used the centrifuge multiple times and no one has ever mentioned different units; as far I was considered, centrifuges used rotations per minutes. The google entries wrote differently. Cells are normally spun at 500 RPF or 1,500 RPM for about five minutes; I spun the cells at 1,500 RPF. Still in denial, I proceeded with adding the dye to check for cell death in my positive control. I had a group of cells that were in media for the entire hour and were supposed to mostly be alive. Turns out, only 17% made it through.

I just couldn't believe what was going on. It took me about four months of hard work and begging to hold on the opportunity to run this experiment and all my cells died because of entering the wrong units? I spent four months testing every chemical and treatment to figure out the protocol. The precious cells that I spent hours skipping (with permission of course) school died without revealing the effects of Caveolin on coping with hypo-osmotic stress.

But with any mistakes, this one made me realize how a little bit of inattention could lead to terrible consequences. Ever since then, I've paid attention to every little detail and asking questions that I once thought were insignificant or stupid. This instance had me depressed and upset for some time, but in the long run helped me become a more attentive intern that asked questions about every little detail and had forced me to reflect on all the skills I have acquired.

I have been with this project from the start, I wrote out the procedure multiple times and tested out every chemical to understand how exactly is affects the cells and the experiment. This internship is my first time working in a lab, and I learned about many basic skill, from pipetting to using big, expensive machines such as the digital light microscope and the centrifuge. Many of these skills were actually acquired through a process of making mistakes and learning from them and for that reason, I doubt I will ever forget these mistakes and what I've learned from them.