Week 8:
Carver College of Medicine

July 15, 2014

We were doing similar experiments this week as the past several weeks to get more of the data and also to increase the reliability. Thus, most of the work this week followed similar procedures as I described before. In the first two days, I continued the nucleus morphology assay and finished counting six more wells. Next week I will be unblinded and finally know if my results actually meet our expectations. I did some poly-L-lycine coating and washing this week, but this time I also washed some 4-well chambers which would be used later for nucleus morphology assays. The washing of 4-well chambers required twice with sterile water with 1 mL in each well, the same amount as each well of the 24-well plate.

I did more luciferase assays this week. I first assayed the Gaussia luciferase (Gluc) for plates 3 and 4, which contained only the secreted Gluc with the media but no cells. I added 25 µL of 2X lysis buffer to each well to maximize the activity of Gluc which would be soon tested. After shaking for one minute, I added 1.5 µL of Gluc to each tube that contained 20 µL of Gluc substrate and measured the luminescence using the luminometer right away since it was a flash-type reaction. We always used a large excess of substrate so that the activity of the enzyme would not be limited.

Plates 5 and 6 still contained the cells which were transfected so that they secreted both Gaussia and Nano luciferases (Nluc), so I did Gluc and Nluc dual luciferase assays on these plates. After washing the plates with DPBS, I added 60 µL of 1X lysis buffer to each well and shook the plates for five minutes. Then I added 1.5 µL of Gluc, which was released from the cells by the lysis buffer, to 20 µL of Gluc substrate in tubes and measured. After measuring the Gluc, I assayed the Nluc by adding 25 µL of Nluc into 25 µL of Nluc substrate in an empty 96-well plate. Then I transferred all solutions to tubes and measured the luminescence.

I also helped out Ron this week with the transfection of neurons in the laminar flow cabinet. I added D, 9A, 9ED, Mas70, A, and VPE transfections to the neurons in the three 24-well plates and two 48-well plates as they were marked. Every well in the 48-well plates needed 25 µL and that of 24-well plates needed 50 µL.