What are the best ways to count dissociated DRG neurons before plating them?
There is a lot of debris in an acute suspension of DRGs so it’s difficult to make out neurons from debris and non-neuronal cells.
Hemocytometers works well for clean suspensions but are difficult for messy suspensions like DRG. I’ve used live dyes like calcein AM before and it helps to pick out cells.
Does your protocol use a 70 um cell strainer followed by a 15% BSA cushion? I find most debris is gone, and the neurons are nice and round so are easy to count. If useful, happy to share my protocol.
Thanks @adgaudet. I would love to see your protocol. We don’t do the BSA cushion, although I know some do. I actually want non-neuronal cells in my cultures, but it would be good to remove them if needed, and debris. a.chamessian@wustl.edu. Or if you’re so inclined, consider adding to the community protocols at Pain Researcher Community Protocols - research group on protocols.io
I want to share some recent updates on this question. I find counting DRG dissociated cells to be difficult without the use of some dye to highlight what is cell and what is debris unambiguously. Furthermore, it is also useful to count how many neurons you have vs non-neuronal cells, rather than just lumping them together and counting ‘cells’.
Calcein AM dye is an easy way to achieve this, combined with disposable slide-like hemocytometers.
Here is a recent mouse DRG culture combining calcein and the C-chip hemocytometer
Very simple. This is a 1:500 dilution of 1 ug/ul calcein solution.
Going forward, this is how we will count our cultures, both mouse and human, to get consistent plating for axon assays.
