Hippocampal Neuronal Culture

Coat MatTek dishes with 1.5mL of PDL solution for 3h 0m 0s at 37°C.

Wash dishes twice with culture grade water and incubate the dishes at 37°C for 0h 30m 0s.

Wash the dishes one more time and let dry. You can keep the dishes at 4°C for few weeks (In this case, seal the dishes with paraffin film).

Just before starting neuronal culture, prepare papain solution and leave at 37°C water-bath.

Dissect hippocampi from P0 (postnatal day 0) new-born mouse brains using a stereo microscope. Collect tissue in ice cold complete HBSS.

Aspirate medium and wash 2-3 times with 10mL fresh ice cold complete HBSS.

Add DNAse (final concentration: 20μg/ml) to papain solution and filter sterilize.

Add DNAse (final concentration: 20μg/ml) to trituration solution and filter sterilize.

Aspirate the enzyme solution and wash twice with trituration solution

Resuspend samples in 1mL trituration solution. Gently dissociate neurons with a P1000 filter tip by pipetting up and down for 10-12 times. Avoid generating any bubbles.

Count the cells.

Seed the cells on the PDL coated MatTek dishes in pre-warmed Plating Media (2mL).

(0.4 million cells / one MatTek dish).

After 3h 0m 0s incubation at 37°C and 5% CO2, change the plating medium to pre-warmed Complete Neurobasal Media.

Remove 500µL of media and add 750µL of fresh complete neuronal media at DIV (days in vitro) 4, 7 and 14. Incubate the media at 37°C before adding it to neurons.