No replies to this topic

[Mighty Mouse]

This is a re-post from the neuroscience forum, but I thought I might get some more info from the folks here in cell biology...thanks so much for reading!

So I have recently been beginning to work with primary neuronal cells (mouse cortex) and am in need of some insight and advice. The cultures had been going along great for a couple of months with only some minor hiccups. I've been optimizing my manipulation protocols etc. and all was well for the most part. However, over the past few weeks I have not been able to get ANY of my four cultures to work (before this I had a very high success rate), and I'm tearing my hair out trying to figure out what is going on.

I dissect out cortex, put in three cortices per plastic tube, treat them with papain for 40 min, wash several times and triturate with fire-polished glass pipettes. I plate 2 million cells per 35 mm dish in neurobasal + B27 + L-glutamine + pen/strep. An hour after I plate I replace the media (and if I have enough hibernate-A I give 'em a brief wash in that to remove any floating debris and dead cells). Lately my neurons show essentially no extension of processes after one day and are basically dead after a couple of days (incubated @ 37C, 5% CO2). Just yesterday I opened a new bottle of media and a new bottle of B27 (which I aliquot into 1 mL aliquots and store at -20C). I even tried plating cells in new media + previous B27, old media + new B27, new media + new B27 in a different incubator. All crapola.

Other things to consider are the L-glutamine (which I doubt, it's stored at -20C and I add it fresh every time I make up media) or the pen/strep, but I don't see how that would stop my cells from growing as there are no obvious signs of infection as best I can tell. One other possibility is that I recently got in a shipment of new sterile plastic tubes (not your typical Fisher 15 mL falcon tubes, but some Sterilin brand tubes w/ a round bottom) I use for the enzyme digestion and trituration steps.

I would VERY MUCH appreciate ANY insight. This is my primary experimental system and things were going so well! I am at a complete loss as to what else there could be! Help!

Thanks

MM