This Tuesday (April 23rd), I went to RPI for another day to set up a new experiment! This experiment is similar in process to the experiment I was introduced to on December 11th. However, in that experiment, we were testing the natural permeability of the blood brain barrier, and this time we're testing the permeability of the blood brain barrier with our manufactured loop-2 protein added! As in the other experiment, the set up is two wells (one inside the other) with a filter on the bottom of the inner one separating the two, as seen in the figure below. On this filter, there is a monolayer of brain cells to represent the blood brain barrier.
We will have many of these setups in order to test solutions with different size molecules. We will be testing solutions with molecules that are 4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa. All of these molecules are dextrans, or different size sugar molecules. For the normal type blood brain barrier that we tested earlier, the permeability to size curve looked like the black line below.With the peptide modulator that we have made, we hope to make the curve look like the red line below.
Today, we made the peptide solution to be added to the cell monolayer. We had to make sure that the protein was at a certain concentration in the PBS solution, so we used a Nanodrop spectrophotometer like the one below. The Nanodrop spectrophotometer analyzes the concentration of the solution by measuring absorption.
We also changed the media of the cells to a non-phenol-red media. The cells are usually cultured using phenol-red media because it is a pH indicator. When the media is exhausted and needs to be changed, the media changes from purple to yellow. However, using this media in our experiment could alter the results. To change the media, we used a vacuum line to remove the old media and then added the new media to both the top and bottom wells. Once all of the materials were ready, we added the peptide solution and dextran solutions to the wells and started the experiment.
I can't wait to see the results!
Kailin, your writing has become so sophisticated! You seem like a graduate student in the lab. I hope that your good work is helping move things along in your project.
ReplyDeleteI am looking forward to the data that you collect!