I want to add calcium imaging (of dissociated neurons in a dish) to my repertoire of skills. I'm seeking a good introductory learning resource that has very practical info to help me execute my own calcium imaging experiments. I can't seem to find anything that fits the bill. There are reviews on the conceptual aspects of calcium imaging, but not so much on the specific details of doing calcium imaging (e.g. what equipment, what dyes, which programs to analyze data, etc.).
Any recommendations would be great. Thanks.
http://www.cell.com/neuron/abstract/S0896-6273(12)00172-9
Hi! I highly recommend the Molecular Probes handbook from ThermoFisher Scientific. Particularly, Ch. 19 outlines a lot of different calcium imaging dyes.
You should look at specific published studies to get idea for specifics in your application, but here is my generic protocol for imaging Ca2+ in primary neuronal culture (I use Fluo-8 AM mostly):
-dissolve the 50ug Fluo-8 in 20% w/v Pluronic F-127 in around 5ul DMSO, to create a stock concentration of 10mM. this can be kept frozen for a long time
-dilute to working solution (media) and incubate: i like to use 5uM, 30 min at RT, but you should work out concentration, time and temperature for your cells.
-wash in dye-free media 2 times, then incubate at 37C for around 30 mins. you will know your wash incubation is not long enough if your baseline signal is increasing over time (this means esterases are still cleaving the AM). if you incubate RT you may need longer times.
-image using FITC/GFP optics!
notes: depending on the type of cell, i sometimes include 0.1mM sulfinpyrazone in the wash and imaging media to inhibit organic anion transporters that kick out the deesterificated dye from the cytosol.
i use ImageJ for analysis, standard dF/F procedure on user-selected ROIs.
please feel free to reach out if you need any more guidance!
Dear Alexander,
As Alberto mentioned, there are different calcium dyes. In addition to the Molecular Probes handbook, you can get helpful tips from Current Protocols in Neuroscience. This chapter has instructions that are similar to Alberto’s, with a helpful chart of some characteristics of a few calcium indicators:
http://onlinelibrary.wiley.com/doi/10.1002/0471142301.ns0203s49/full
You might find the “commentary section helpful, since it gives more background, equipment basics, and troubleshooting hints.
The lab I work in also keeps a copy of an older volume of Methods in Cell Biology (Volume 40) because it has some proactical hints for the older dyes as well as for manipulating calcium levels in the bath and in the cells. It has since been updated, and you might be able to get a copy from your institution’s library or thru ILL:
https://www.elsevier.com/books/calcium-in-living-cells/author/978-0-12-374841-6
Good Luck!
Jill
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