3T3-L1 detach after 100% confluency

Hi, I am doing 3T3-L1 differentiation study now. One big problem is that after cells grow into confluency,  these cells become very easily detached during medium change on 24-well plate, especially in the central area of the plate. Even I was very careful and slow when pipetting the medium to avoid shearing force, for some wells there were still holes in the central area. I use Costar polystyrene plates. I wonder if this is a common problem for confluent 3T3-L1. Is coating the plate with laminin or some other coating agents helpful to overcome this problem? Also I notice that my cells are kinda easy to detach during subculture, only 1min in Trypsin-EDTA, most cells will come off. Is this normal for such a adherent cell line? Thanks.

 Hi ljbio,

Yes, all the issues that you touched on are commonly encountered while culturing/differentiating L1 cells. I never tried to use coated plates, and not aware of others who do that. 

One piece of the comment- while changing the medium, do you use a vacuum-based method to remove the media? If yes, try to manually pipet out the medium. Also, set the force of your pipettor at the lowest setting as possible, and alway remove and add the medium from the side instead of from the center of the well. You can even mark a corner so that you can pipet out/in from the same spot every time. If I remember correctly, changing medium on Day 2 and 4 during the differentiation is the most challenging part as the cells start secreting tons of materials into the medium and make it sort of sticky- This is when you need to remove the spent medium as slow as possible....

As for the trypsinization for subculturing, yes, they are easily detached. I used to add 0.05% Trypsin-EDTA, and pipet up and down few times and skip the incubation all together...

Good luck.

Hi Pippuri, thanks a lot for you reply!!  This is really helpful! I do pipet manually, but as you said, maybe I used too much force. One problem is that if you do the pipetting very very slowly, for 24 wells or even more, it would take very long time (about 10 to 15 miu for 48 wells). I worry whether this long time period in RT(25 ^oC) will have any harmful effect on the cells. Thanks.