Hi guys,
I am trying to establish conditions and choose the machine to be able able to measure baseline and stimuli induced calcium oscillations in primary neuronal culture. I tried Cytation from Biotek company, however, I got a false positive signal because of mechanical artifact after injection. Basically I saw a response even after vehicle administration. I also do not see consistency in baseline Ca oscillations between wells. Any idea what are the best conditions to see consistent oscillations? Also, I am using FluoForte AM from Enzo. ANother question is whether it is crucial to remove the dye before measurement, since the manufacturer protocol says the dye should stay. However I have variability in fluorescence between unwashed and washed wells with neurons. It seems that this methodolody is quite sensitive for details in culturing and measurement. Any advices or experience? Especially that mechanical artifact after injecting the drug makes troubles since it is hard to evaluate whether the response is real or false positive. Also, what machine other than FLIPR or FlexStation do you recommend and what setting (injection speed, volume, etc.)? Thanks.
I cannot answer all the questions you posted, but at least can address one.
Since you are using the AM version of the dye, it is imperative that you wash it from the solution for a least 30 minutes before you start measuring calcium. This is often called the deesterification step during which the AM is cleaved from the flurophore and now (and only now), the flurophore is sensitive to Calcium.
Are you using a plate reader?
Thanks Oliver, I was testing Cytation 5, Biotek plate reader to see whether the machine is capable doing it. However, the injection speed setting range from 235-300 ul per second, thus I suppose that is the reason of false positive results in fluorescence change with vehicle. I got the same response even after washing out the medium. prior to measurement. What equipment do you use and what settings?
Hi Marcela-- I did calcium imaging in rat and human neurons a while back, using a BD Pathway high content imager. There was a brief flash of light at injection, but we were able to minimize it by keeping the injected volume small relative to the volume of ACSF on the cells.
what was the injection speed you used, Richard? are there any other problems you had to deal with while optimizing the conditions? anything regarding neuronal culture? It seems I do not see baseline oscillations in every well, it is rather random phenomenon. What is you experience?
Hi Marcela-- we added the drugs at 20-40 microliters/second. I think my experiments were a bit different than yours, since we were using the Pathway to record fluorescence in individual neurons; I assume the plate reader is averaging the whole population?
I recall that it took about 3 weeks of culture to see spontaneous activity synchronized across most of the rat neurons. Before 3 weeks, not much happened until we disinhibited with picrotoxin.
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