I am trying to polarize murine raw cells into 9 wells (C=6*10^4 and passage=7) with cytokines. I have used LPS and IFN-gamma for M1 and IL-10 and IL-4 for M2. But after polarization, the density of cells at the edge of wells is super high, especially for M2. Does anyone have any idea about this?
Hi Arezoo,
When using culture plates, especially multiwell plates, there is a tendency to employ very small volumes of culture medium or buffer. This may cause the liquid depth around the edges of the well to be relatively much thicker than that in the center of the well due to attractive forces between plastic and water. This can result in a very uneven cell distribution such as the one you describe, few cells in the center and many in the periphery of the wells. The simplest solution to this is usually to increase the buffer volume by 2-5 fold in each well to diminish the impact of this edge effect. If this is not practical, then other means of reducing the meniscus thickness can be tried.
This edge effect will have its biggest impact while cells are plating down and adhering to the bottom of the well. After adhesion, cells will not usually move around much. So, use larger volumes when cell attachment is occurring and then smaller buffer volumes can be used in later steps for valuable reagents.
I don’t know if this will resolve the problem you described, but it might help and it’s easy to test.
Good luck,
RW
