Recently, I am a novel in neuron field and use the primary cortical neuron for research. But the primary neuron show the low survival rate and unhealthy, I don't know how to improve the skill or same tips during the experiment. The procedure following:
the dish coat the poly-L-lysine(0.1 mg/ml) for overnight
Dissect E16.5 mouse brain (Hippocampus and cortex) in Ca/Mg free HBSS on ice.collect the dissected tissue in the 15 ml canonical tube and wash with HBSS for 3 time and centrifuge 1000xg for 3 mins in 4 degree.Add 500 µL 0.05% Tryspin (0.5 % EDTA and Tryspin) and 500µL HBSS in the sample and put in the 37 °C water bath for 3 min.Add 10 ml NeuroBasal Medium with 1% FBS to neutralize and wash for three times(centrifuge 1000xg for 3mins at4 degree).Add 10 ml NeuroBasal Medium with 1% FBS to neutralize and wash for three times(centrifuge 1000xg for 3mins).lock (ps. No bubbles during triturating).Keep the sample for 1 min for settling tissue blockTake supernatant (ca. 4.5 ml) for another new 15 ml canonical tube and centrifuge 1000 rpm for 5 min at 4 degreeAdd 10 ml HBSS for wash two times and centrifuge 1000xg for 3 mins Resuspend the cell pellet in NB medium with B27 and L- glutamine (Cortex about 10 ml and Hippocampus about 1 ml)Seed the cells on the vessels and replaced half medium for 2-4 daysAfter all the procedure, measure the dead and survival cell number. The overall survival is around 40-50%.
Also, i took the image at the DIV 5. They got many dead cell ( the light buds) and hard to remove in the plate. How to optimize the primary cortical neuron culture and minimize the cell death ?
