Most protocols use one or more proteolytic enzymes in the dissociation, plus DNAse to break down chromatin that can trap cells and cause clumping. These papers and those they cite, give a good overview. The dissociation protocol starts about halfway through the second paper.
Article Dissociation of neonatal and adult mice brain for simultaneo...
Article Culturing pyramidal neurons from the early postnatal mouse h...
In my experience trypsinization is very important. After isolation of cortices I trypsinize for 15 - 20 mins at 37 C and then stop the enzymatic digestion by adding full media (containing FBS). After spinning the cells down (1500 rpm 3 min) the pellet is resuspended in fresh full media and passed through a cell strainer.
At times when I have not trypsinized enough and/or not triturated/resuspended enough the cell yield is very low. If I have been caught without a stock of cell strainers clumps of tissue can be seen in the culture and this negatively affects the culture.
Ellen A G Chernoff thank you so much for these papers. Very helpful.
Steven Fagan I did another culture yesterday and followed these tips, was looking much better under the scope and when i counted. thank you.
Kavitha Jade Brunner that's great to hear! Good luck with the cultures and let me know if I can help with anything further.
Kavitha Jade Brunner, how many days after the culture did you take a look at the cells..?? In general, the mixed cultures come with a lot of debris in the first 2 to 3 days. We generally wash the cultures and supplement fresh media for 2 days after the culture. Cells begin to look much ``cleaner`` after 5 to 6 days of culture.
Also, we trypsinize the cell suspension for 10 mins with a brief vortexing step after the first 5 mins. Then homogenize it after adding with DNAase. In our experience, passing the suspension through strainers tend to reduce the yield.
Phani Sankar Potru I examined the cells immediately after plating the single cell suspensions. Do you change the media every day for the following two days after culture? How often do you recommend changing media?
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