Progress….   Leave a comment

Things have gone better in the lab this week. For the past month or so, we’ve been working on getting proposals submitted by deadlines. I’ve noticed that of the total amoiunt of time spent writing a proposal, over half is spent on non-science forms: budgetary forms are a big chunk, but there’s a large number of non-science documents that have to be generated.

Anyhow, now that *that’s* done, we got back to work. We’ve been working on getting Fura imaging working, and there are a lot of different technical details that have to be resolved: loading the cells with enough dye, but not toxic amounts; keeping the dye in the cells; and this week we worked on keeping the cells alive under the microscope. As written previously, we’ve had problems keeping the cells happy for more than a few minutes- there was a bacterial infection, the cells detached from the permeable support, issues with CO2 regulation in the incubator, problems with the automated image acquisition routines, etc.

This week, we were able to keep the cells alive and happy for up to 6 hours under the microscope. Even better, the image routines now adjust to keep everything in focus over the entire experimental run. This gives us confidence to try a test run, loading the cells with Fura, putting them under the microscope, and using a positive control (ATP) to elicit a calcium signal. We’ll give it a shot tomorrow, hopefully all will work well.

Now, the reason we are doing this is because a spike in calcium concentration is symptomatic of a cellular response to a stimulus. That is, calcium imaging is used to decide whether or not the cell responded to a stimulus we experimentally applied. The stimulation we apply is via laser tweezers or fluid flow. The first one we will try is laser tweezers. But first, we have to make sure the Fura imaging works properly.


Posted November 3, 2010 by resnicklab in Physiology, Science

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