3T3-L1 cells peeling/curling when differentiated - any help? - (Sep/07/2008 )
Hello,
I am working with 3T3-L1 cells and I've been having a problem on and off that I was wondering if anyone can help me with. I am differentiating 3T3-L1 cells with your standard induction cocktail. A day or two after I change the media from the induction cocktail to growth medium + insulin, I notice my cells start to peel at varying degrees from the wells. I am using standard 6 well plates (not collagen coated) and I've been having this problem for a little while now. One experiment might be ok and the next, I will get complete peeling of the cells. The cells do not appear to be peeling due to toxicity. Anyone have any insight?
Thanks!!!
3T3-L1 are really sensible to passage number. If you started from ATCC lot, I do not recommend more than 10-12 passage. Cells will peel off like you described and cells won't differentiate.
Make sure to have a sufficient stock of cells and try not to work with old cells.
Also, these cells are sensible to contact inhibition. So if they reach confluency while you passage them, they will peel off also. Its a tricky cell line!
It has been quite some time since I've worked with this cell line but I looked back through my protocol notebook and found my protocol for differentiating L1s.
1. grow cells to confluency
2. replace media on cells (10% calf serum)
3. next day, replace media with differentiation media
4. change media in 3 days to DMEM (10% FBS)
5. refeed every 7 days.
I don't remember why the calf serum is important but I remember a big deal being made of it. Also, I don't think one or two days is enough for differentiation. I'm wondering if the variation you are seeing is the difference in time you are allowing for differentiation. You said you differentiate for a day or two but this is a very important parameter and needs to be kept constant from one batch to the next in order to get reproducible results. Otherwise, I remember these cells as not being very adherent and you have to be very careful to pipette media/pbs down the side of the well and not directly onto the cells.
1. grow cells to confluency
2. replace media on cells (10% calf serum)
3. next day, replace media with differentiation media
4. change media in 3 days to DMEM (10% FBS)
5. refeed every 7 days.
I don't remember why the calf serum is important but I remember a big deal being made of it. Also, I don't think one or two days is enough for differentiation. I'm wondering if the variation you are seeing is the difference in time you are allowing for differentiation. You said you differentiate for a day or two but this is a very important parameter and needs to be kept constant from one batch to the next in order to get reproducible results. Otherwise, I remember these cells as not being very adherent and you have to be very careful to pipette media/pbs down the side of the well and not directly onto the cells.
Thanks for your help Madrius and rkay447!
Just to clarify, this is what I do:
1. Plate 100,000 cells into 6 well plates
2. Grow to confluency (approximately day 3-4) with 10% FBS
3. Induce cells to differentiate (growth medium + 10% FBS + IBMX/insulin/dexamethasone) (day 0)
4. Change to growth medium + 10% FBS + insulin 10 ug/ml on day 2
5. Stain cells anywhere from day 7-9.
In all of my experiments, I get great differentiation and lipid accumulation. The problem is the peeling which happens seemingly randomly - sometimes on day 3, others at day 4 or 5. A few times, I got no peeling at all and everything worked great! When I get peeling, it usually isn't the entire monolayer lifting off the wells - it's usually a piece of the monolayer here or there...sometimes with random "holes" in the center of the monolayer. It doesn't appear my conditions are killing off the cells (i.e. toxic) - it just seems as if they lose their ability to adhere to the wells.
Has anyone tried collagen coated wells? Any other thoughts?
Thanks everyone!
I know what you mean by "peeling off". And as I experienced it, the cells won't differentiate if the well/petri dish peels.
And I use the exact same protocol as yours. Try working with "young" cells. Since i've noticed old cells were more susceptible of peeling, i've been using only fresh cells, and I've never experienced peeling again.
And I use the exact same protocol as yours. Try working with "young" cells. Since i've noticed old cells were more susceptible of peeling, i've been using only fresh cells, and I've never experienced peeling again.
Good to know. Thank you. For my next round of experiments, I'll try a lower passage number. What passage number do you stop at? You previously mentioned 10-12. I'm currently at 15. The cells do start to differentiate but then peel off after changing the induction medium.
Exactly.
Try "younger" cells.