Help! neuronal cell culture problem

Don Cates cates at cc.umanitoba.ca
Thu Aug 3 14:39:54 EST 2000


On Thu, 3 Aug 2000 13:50:35 -0500, "Serotone"
<serotone at replies_to_newsgroup.com> wrote:

>"Don Cates" <cates at cc.umanitoba.ca> wrote in message
>news:39881d93.49487258 at news.cc.umanitoba.ca...
>> I've been doing cell culture of dissociated neurons from fetal rat
>> medulla for several years. Recently the wheels have fallen off.
>> I use 35 mm plastic dishes with a collagen (Vitrogen) coating. I then
>> plate with neuron free medullary background cells for a week or more,
>> then plate with my dissociated cells (mince with scissors, treat with
>> trypsin for 15-20 min, gently triturate). The last few times I have done
>> this, the cells seem to lift off most of the dish and form long, thick
>> 'ropes' of cells which include the collagen and background cells. I've
>> replaced my various media and components with fresh material with no
>> luck. It is not invariable, I get the odd good dish.
>> Can anyone suggest anything?
>
>Do these ropes consist of lysed cells or healthy ones?  Trypsin activity
>remaining in the cells suspension would cause this.....

Healthy cells. I thought of that early but since the 4ml of 0.125 mg/ml
trypsin is passed through 10 ml of MEM with 5% FBS I thought that it
would be inactivated. But that was a while ago and nothing else has
worked so I'll check it again more thoughly.
--------
Oh my, I feel dumb! I just rechecked and the stock trypsin that I got
from a new supplier is 10x the concentration that I was using before. So
I have been using 1.25 mg/ml trypsin. That might be it.

Thanks for the prod to go back over the obvious.

Don Cates
cates at cc.umanitoba.ca






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