Week 9:
Carver College of Medicine

July 22, 2014

This week I finished my final set of dual luciferase assays with the Firefly and Gaussia luciferases. Usually we would do the dual assay with NanoLuc and Gluc since Fluc needs ATP in the buffer to work. Fluc was also larger in size, which meant that the cells would use up more energy in producing these enzymes. This time, however, we made an error by misreading the label of the plasmid and ended up with cortical neurons transfected with Fluc and Gluc. Anyway the experiment should still work since Fluc served the same function as Nluc by indicating the relative number of living neurons. Moreover, we were using a new model for stroke this time instead of the OGD and glutamate treatments that we normally did. This model was called the chemical-induced oxygen-glucose deprivation (cOGD), in which we treated the cells with 10 mM of 2-deoxyglucose and NaN3. The experimental procedure here was still the same as any previous luciferase assays, except that we added a stop and glow buffer in the middle to stop the enzymic reaction with Fluc and start the one with Gluc.

The results were shown in the slide below, which was taken from my final lab meeting presentation. We could observe that the results of the cOGD model largely agreed with our hypothesis except for the Mff-D transfection, which was supposed to be the control but ended up abnormally high in survival rate. Anyway we would redo this experiment with Nluc / Gluc combination since we knew that it would probably produce better results.

This week I was also busy preparing for my final lab presentation, which would be next Tuesday. I finished a preliminary draft of the PowerPoint which was also attached in this blog post.