Today was a funny day. My mentor while showing me how to collect for protein accidentally spilled some of the cell sample. Later on, I added 0.5 uL instead of 5 uL of a dye while running the mitopotential assay. The assay still worked though, and I got some interesting, significant results with neurotransmitter and Bortezomib treated cells. Of course, the dead cells could have been underestimated, but at least I think that this experiment might not be such a shot in the dark anymore.
I pre-treated cells with c-Myc inhibitors and later treated them with Bortezomib to monitor the effects on Bortezomib-induced apoptosis. I plan to collect for protein tomorrow and run a Western blot on Monday.
I ran the XTT assay and got some not so interesting results. Bummer! That’s how research sometimes goes… I also plated an experiment to run a mitopotential assay; hopefully, those results on Friday will be more interesting.
Today, I treated HeLa cells with a peptide and Bortezomib. I had fun again using the multichannel pipette and can’t wait to run the XTT assay tomorrow and see the results!
I made cDNA from mRNA today. It was an interesting, fun process, but I liked setting up for an XTT assay and plating HeLa cells even better. Multi-channel pipets look so cool and are even more awesome to use! They also get me thinking about those precise, fast robotic pipetters.
RNA isolation is interesting! The most curious part is how the liquid will separate into 3 layers. The two most important things are to be cold and clean with RNA; my mentor emphasized these two a lot! We also imaged a Western blot, which contained the results of the ubiquitination upregulation experiment. Apparently, the Bortezomib combination with increased ubiquitination upregulates apoptosis. The search is on to find out why!
When everything heads south, what do you do? I was asking myself this question a lot today; the cells were dying. There was no autoclave available at the moment, and it had seemed like we’d tried everything! We just couldn’t figure out why the cells were dying. We decided to thaw some HeLa cells to experiment with in the near future if things worsen.
Today was quantitative PCR day! Plating 45 wells was a little annoying at first; there was so much pipetting! In retrospect, however, it was good practice, and the results turned out to be pretty good.
I plated cells to further explore combinations of Bortezomib, D3 pathways’ upregulation, and Gli1 pathway inhibition on multiple myeloma cells (side note: Hedgehog activates the Gli1 pathway). I also pre-treated cells with ubiquitination enhancers for the experiment that I started yesterday. It’s fun working on multiple experiments at once; it’s not something I’m used to doing. However, when working all day in the lab, I realized the importance of planning carefully to make sure that I’m not waiting around for one experiment to finish but am not overstretched either.
I started experimenting with ubiquitination enhancement and Bortezomib-induced apoptosis in general. I plated the cells under the hood today. The results should be pretty interesting!
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