Cell Swelling Assay: My first experiment

I admit that I haven't blogged a whole lot about my summer experience at the Patel lab, but that doesn't mean I didn't learn enough. In fact, I go on for about a few hours talking about all the new techniques, assays and experiments I've seen and my million blunders and mistakes.

A few weeks into summer Dr. Jamila Siamwala approached me and asked me if I could count some cells for her. Not sure of this task entailed, I was glad to be given some work or a task I would be responsible for. I had to count the number of alive cells and the number of dead cells (were colored blue). After what seemed like hours of work split over the course of a week or two, I finally finished and presented the data to her. We were excited to find a significant difference between the treatment groups. Then, Jamila asked me to run this experiment with a few modifications.

The goal of this experiment was to see if there was a difference in cell death due to hypo-osmotic stress between dermal fibroblasts with and without a caveolin mutation. Caveolin is a protein that plays a major role in cell signaling, fat storage, endocytosis and the production of caveolae. Caveolae form mini invaginations in the plasma membrane and serve as a mechanism that allows cells to expand. My goal is to see if mutated caveolin inhibits the function of caveolea. This will be measured by assessing cell death.

Methods:

A 24 well plate was set up as the following picture:

Cells in the wells labeled H2O2 were placed in a one millimolar hydrogen peroxide diluted in MEM media for ten minutes and cells in the wells labelled hypo-osmotic media were placed in in a 30 osm (nine parts water to one part media) buffer for 30 minutes. Cells were then lifted using Trypsin and stained with Trypan blue for five minutes. They were then analyzed for cell death using a hemacytometer. All solutions were collected and combined with the respective wells they came from so that lifted cells could also be accounted for.

But please, do not be followed by this one paragraph procedure paragraph. The following document shows the timeline of this experiment.