Week 7 - Flow Cytometry Failure

Once again, I worked on many different projects this week. Every day I come in I never know what I’ll be working on since Rachel and I have so many different experiments we want to run. On Monday and Tuesday I mostly worked with the flow cytometer. I made a new calibration curve for T. weiss to compare to the old one, and it was very different. The slope for one line was positive and the other one was negative. I ran a test with randomly made buffers to see which line would be more accurate but they were both terrible. The second one I made was off, but the first line was extremely off. It gave me a pH value of 16 when the actual pH was 6.7. 

Rachel and I decided that we would not use the flow cytometer to measure intracellular pH because it’s values could not be trusted. We started some 24 hour pHstat experiments and used the spectrofluorometer to measure intracellular pH instead. It worked well although the process took longer than the flow cytometer. We had to keep redoing the experiment however because something always seemed to go wrong. The first time the computer program had an error in the middle of the night and the second time the pH range was accidentally set to be too big so the program didn’t work well. Finally our experiment on Friday went well and we got usable results. One of the results that was interesting was that the intracellular pH was consistently lower than the media’s pH.

The hardest part of this week was running an experiment to show which incubation time for SNARF was the most efficient when using the spectrofluorometer. I used T. weiss. and prepared my samples the same way as when I test their intracellular pH, except I had different incubation times of 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 6 hours. Normally I use an incubation time of 30 minutes so my results should confirm that it is most efficient. It was a lot of work to test because my samples were always ready at different times. I was constantly running around trying to measure some on the spectrofluorometer while continuing the preparation for others. At some point next week, I will probably run the same experiment with Alexandrium to see if the results change at all.