rat primary cortical neurons - early death in culture (Jul/13/2010 )

hi all,

i m having hard time to get my rat cortical neurons survive. They look great until 4 days or so, than they start to get deteriorate, losing their neurites and look bubbly and eventually die around day 5 or 6. never survived more than 7 days yet.
This is how i culture;

Dissection on Day 16 or 17, done in HBSS buffer on ice.

Trypsinization; 0.25% Trypsin for 20 min at room temperature. Wash 3 times with MEM (5%FBS, 20mM Glucose, 2mM Glutamine) at RT.

Trituration in NBM with B27 , 0.4mM Glutamine, 20uM Glutamic acid ; with 10ml pasteur pipette for 20 times or so. They dissociate very well without any struggle.

Let them sit for 10 min and remove the supernatant (cell suspension). Count and plate with the same NBM (as above), at 1,000,000cells/ml. Plates were coated with Poly-D-Lysine (40ug/ml) for 2-3 hrs at 37C.

Change half the media at DIV3 with NBM+ B27+ 0.4 mM Glutamine (no more glutamic acid).

Any help or suggestions would be highly appreciated; i m trying to solve this problem for months now...

Thanks!!

Try different Trypsin.. I know we had a lot of issues when we changed even the batches from the same company. I agree, to work with primary neurons.. its a pain.

Try using glutamax instead of glutamine, since it the last spontaneuosly breaks down in the medium and creates toxic products. Glutamax is more stable. Don't change your medium on neurons COMPLETELY, only half.

Inaddition to other suggestions, tritration can make a difference. Donot tritrate the neurons harshly or close to the conical end.

Do you do cortical neurons from day 16-17 postnatal? have you ever tried it in mouse?
someone uses papain instead of trypsin because it is more gentle...but if they do not survive cant it be because of the coating? what P-D-L do you use? low MW is not too good...and wash it well afterwards..

marco

gengoz on Tue Jul 13 14:40:33 2010 said:

hi all,

i m having hard time to get my rat cortical neurons survive. They look great until 4 days or so, than they start to get deteriorate, losing their neurites and look bubbly and eventually die around day 5 or 6. never survived more than 7 days yet.
This is how i culture;

Dissection on Day 16 or 17, done in HBSS buffer on ice.

Trypsinization; 0.25% Trypsin for 20 min at room temperature. Wash 3 times with MEM (5%FBS, 20mM Glucose, 2mM Glutamine) at RT.

Trituration in NBM with B27 , 0.4mM Glutamine, 20uM Glutamic acid ; with 10ml pasteur pipette for 20 times or so. They dissociate very well without any struggle.

Let them sit for 10 min and remove the supernatant (cell suspension). Count and plate with the same NBM (as above), at 1,000,000cells/ml. Plates were coated with Poly-D-Lysine (40ug/ml) for 2-3 hrs at 37C.

Change half the media at DIV3 with NBM+ B27+ 0.4 mM Glutamine (no more glutamic acid).

Any help or suggestions would be highly appreciated; i m trying to solve this problem for months now...

Thanks!!