96W1E

It's been so long since I've been at my internship! It was a good thing I didn't forget what I had learned over winter break, because we started a much more complex experiment today! Today I got to work with a 96 well electrode known as a 96W1E. This wellpad works the same way an 8 well electrode would, but looks quite a bit different! Here is an image of my 96W1E, all filled up and ready to be placed into the ECIS station:

In today's experiment, we used two cell types: MDCK and BSC-1 cells in a dilution series. Dr. Keese demonstrated how to work with a 96 electrode, and proper pipetting techniques using an 8 tip pipette. He first pipetted 150 µl of cysteine into the first six columns of the wells. Cysteine is an amino acid that reacts with the gold of the electrodes, allowing proteins cells to attach and spread easily. While we waited for the cystine to react with the gold, a process that takes at least 5 minutes, he began to treat the MDCK cells with EDTA and trypsin to get the confluent layer of cells to be free-floating. Setting those cells aside, we began to prepare our dilution series. The main idea behind a dilution series is to examine how cells grow in environments of varying sparsity. We created cell mixtures in six test tubes with the following cell compositions: 1x, 1/2x, 1/4x, 1/8x, 1/16x and medium (0x). We got these mixtures by filling each test tube with 3mL of medium (except the 1x tube). The 1x tube was 6mL of a suspended MDCK cell mixture. We took 3mL from this tube and dispensed it into the second tube- 1/2x. We mixed the cells and the medium using a titration technique (aspirating and dispensing the mixture over and over again to make sure the cells are uniformly distributed). This step was repeated for each of the subsequent tubes, therefore making each tube half as concentrated as the preceding tube. Dr. Keese then pipetted the mixtures of varying concentration into 6 of the 12 columns. I repeated these steps on my own with the last 6 columns, using BSC-1 cells. Here is an image that shows the layout of the 96 wells:

Finally, we were ready to place our prepared 96W1E into its station! This station looked different than those that I have used with 8 well electrodes. Instead of teeth that attached to one end of the electrode, this station uses what Dr. Keese called, a "bed of nails" to transfer signals from the electrodes to the ECIS machine. Each of the gold "nails" would connect to a single electrode, relaying the electrical signal to the more advanced ECIS machine, the Zθ. This machine is different than the Z (which I am used to working with) because it uses a "complex impedance spectrum", meaning it can read cell behavior in Z, R, and C (impedance, resistance and capacitance). 

Here is a sneak peak of the first 15 minutes of my graph. Looking forward to seeing my results next week!