Hello, my name is Alp.

I am culturing primary cortical neurons from E18 rat pups. For this I am using a Plating media that is contains ß-merchaptoethanol, L-Glutamine, Glutamate, P/S, Neurobasal medium and B27 supplement. I'm using 0,5 ml 1X Trypsin in 5 ml solution for dissociation. After that I am triturating with 10 ml glass pipette and 1000 ul Polypropylen pipette for further dissociation. I am seeding cells in density of 8-10 * 104 cell / cm2 in PDL (50 ug/ml concentration) coated 60 mm polystyrene petri dishes. I am changing 50% media in 3rd day with AraC media to eliminate glia cells. In the 4th day I am changing 90% media with fresh Neurobasal/B27. Every 3-4 days I am changing media with fresh one.

After this protocol, my neurons seemed okay for 8 days (I added some photos of my neurons). Then, I needed to relocate my cells to 96 well plates for my experiment. For this purpose, I treated my neurons with 0.025% Trypsin - 0.01% EDTA solution for 5-10 minutes to detach and I transferred detached neurons to PDL (50 ug/ml concentration) coated 96 well plates for the density of 104 cells / well ( in 200 ul media). After this process I lost all of my cells.

1) Do you think my 8th day cells look okay? Is there anything wrong? If there is what can I change in my technique?

2) I need to work in 96 well plates. Do you think primary neuronal culturing in 96 well plates okay? For the best results what should I do?