I have tried many times protocol very similar to Brewer 2007, but still I have very low amount of neurons and more glial cells.
I use Hibernate A +B27+glutamine for working, Neurobasal A/B27 medium for culturing, papain for digestion, DNAse and papain inhibitor after digestion. I triturate the cells by glass pipette very carefully, I also tried various gradients...I tried work on ice or on 37 °C/RT, for cover slips I use laminin/poly-d-lysine but nothing much helped :-(
What do you think is the most important thing to have more neurons? Do you have some good protocol for it? Thanks a lot!
Hello,
it would be helpful to know at which point you lose cells.
Do you get low number of cells when you count them before plating? --> Most likely need to optimize dissection protocol
Few neurons attach? --> Most likely need to optimize coating
Do cells attach but not survive/develop? --> Most likely need to optimize the culture medium/make sure all reagents are OK
Do astrocytes proliferate? --> Your culture medium should not promote astrocyte proliferation but maybe you could try to add reagents to block it
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