Using Labtek for Bone marrow macrophages - (Dec/11/2012 )

Hello!

I am using the Labtek slide chamber (Nunc) for bone marrow derived macrophages cultivation and later infected with Leishmania.
I found the cells did not adhere evenly on the surface of the wells. They were on the edge of the well, instead on the center! (I hardly found any cells in center....)
Besides, I tried to infected the macrophages with Leishmania (a free-swimming protozan parasite). The Leishmania just sticked tightly to the surface of the Labtek. And it is impossible to wash them away with simply pipetting....

So my questions are:
1. How to let the macrophages adhere to the Labtek surface evenly???
2. How to wash the wells efficiently, so that uninternalized parasites can be washed away?

Would be anyone here has similar experience to share or give me some advice?? Please please help me!

Thanks a LOT!!

poor cremebrulee

I can't give you advice on the the Leishmania, but i can on the cells.

It is a common problem with cell culture for cells to adhere near the edges of the culture surface, this is called an edge effect and is the result of surface tension of the medium interacting with the wall of the chamber. There isn't much you can do about this other than to add a bigger volume of medium or switch to bigger chambers. It may also be that the cells don't adhere well to the slide, in which case you can coat the wells with attachment factors such as collagen or fibrinogen. You can also buy these pre-made but they are quite a bit more expensive than the already expensive culture slides. Note that you can also do this on glass coverslips in wells.