Protocol for hippocampal neuronal cultures
Andrés Guillén-Samander, Pietro De Camilli
Abstract
This protocol details the procedure for preparation of neuronal cultures from mice hippocampi as it was performed in http://doi.org/10.1083/jcb.202010004 http://doi.org/10.1083/jcb.202010004 but can also be used to prepare cultures of cortical neurons.
Attachments
dn3ubgtzx.pdf
Steps
Protocol for hippocampal neuronal cultures
Coat MatTek dishes with 1mL per dish of 0.1mg/mL Poly-D-Lysine (Sigma) for at least 1h 30m 0s to 0h 30m 0s at 37°C.
Wash dishes twice with culture grade water and let dry.
Prepare papain solution and leave at 37°C for 0h 30m 0s.
Dissect hippocampi from at least 3 P0 mouse brains using a stereo microscope. Collect tissue in ice cold Neuronal HBSS (nHBSS).
Transfer isolated hippocampi into a fresh cold nHBSS containing dish.
Cut tissue into ≈1mm3 pieces and transfer into a 15mL Falcon tube with 10mL ice cold nHBSS and let sediment 37On ice.
Aspirate medium and wash 2-3 times with 10mL fresh ice cold nHBSS.
Add DNAse to papain solution and filter sterilize. Incubate tissue prep with papain solution for 0h 30m 0s at 37°C on a rocking platform.
Aspirate the enzyme solution and wash twice with plating medium and then twice with nHBSS.
Allow debris to settle for several minutes and collect supernatant.
Resuspend samples in 2mL cold nHBSS. Gently dissociate neurons with a P1000 filter tip by pipetting up and down for 10-12 times.
Count the cells.
For imaging, seed 75,000 neuronal cells as a drop (usually around 100µL) in the PDLcoated coverslip of MatTek dishes in Plating Medium.
After 3h 0m 0s to 0h 30m 0s incubate at 37°C and 5% CO2, change the plating medium to neuronal medium.
Remove 500µL of media and add 1mL of fresh neuronal media every 3-4 days.
