Primary neuronal cultures
Itika Saha, F. Ulrich Hartl, Mark S. Hipp, Miguel Da Silva Padilha, Irina Dudanova
Abstract
This protocol describes the preparation of primary neuronal cultures from E15.5 CD-1 wild type mouse embryos. Experiments involving animal models must be performed in accordance with relevant institutional guidelines and regulations.
Steps
Sacrifice pregnant female mice by cervical dislocation.
Remove the uterus from the abdominal cavity and place into a 10 cm sterile Petri dish on ice containing dissection medium, consisting of Hanks’ balanced salt solution (HBSS) supplemented with 0.01 M HEPES, 0.01 M MgSO4 and 1% penicillin/streptomycin.
Isolate each embryo, decapitate the heads, remove the brains from the skull and immerse in ice-cold dissection medium.
Dissect cortical hemispheres, and remove meninges under a dissection microscope.
Collect the cortices in a 15 mL sterile tube and digest with 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 15 μL 0.1% DNAse I for 20 min at 37 °C.
Stop digestion by removing the supernatant and washing the tissue twice with Neurobasal medium (Invitrogen) containing 5% Fetal Bovine Serum.
Resuspend the tissue in 2 mL Neurobasal medium and triturate to achieve a single cell suspension.
Spin cells at 130 x g, remove the supernatant, and resuspend the cell pellet in Neurobasal medium with 2% B-27 supplement (Invitrogen), 1% L-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen).
Plate cells at desired density in dishes or coverslips coated with 1 mg/mL poly-D-lysine (Sigma) and 1 µg/mL laminin (Thermo Fisher Scientific).
