Hi all - first time poster! Hope I can get some good feedback. I am Ph.D. student and am working with an HN2-5 cell line (murine hippocampal neuron). I've successfully done cell culture before and with the same experiment, but for some reason I'm having trouble keeping my cells viable. We do EtOH research and so I have 4 groups - Control, DHA, EtOH, and DHA+EtOH. I am using 12 well plates coated with Poly-L-lysine and seeding at 55,000 cells per well. After reaching 70% confluence I treat with 5uM retinoic acid to differentiate. After 24 hour treatment I begin my EtOH treatment which lasts 4 days. Each day media is replaced with fresh media. I grow cells in 10%, use 1% FBS while differentiating with RA, and 0.5% for treatment. This is per a protocol a lab member of mine used in the past that was successful with these cells.
I have done this experiment on 100mm petri dishes successfully. Maybe a month ago, but now the cells are clumping up on the last two days of treatment (in any group including control), they are shriveling up and there looks to be a good amount of cellular debris from dead cells.
Any idea as to what could be going on?
Dear Jennifer, can I ask you how many times did you observe these events (clumps formation, cell death, etc...) in your cultures? If those occurred just once, I would try to simply look at the common reagents between your experimental conditions (medium, supplements, etc...) and try again with fresh ones. If instead you got these problems more than once, I would hypothesize that you are plating your cells at too low density, and this could definitively account for the signs you describe in your post.
Hope this helps,
Federica
Check plating your cells again in 100 mm petri dishes simultaneously while plating in 12 well plates coated with Poly-L-lysine, and see if your cells are doing ok or not in petri dishes this time. This will indicate if your reagents are to be blamed or not.
did you coat the petri dishes also with Poly-L-lysine as you did your 12 well plates?
- Badges
- Science method