5 replies to this topic

[TracyDuke]

Hi All, I need to do coculturing in the near future, which I have 0 experience on. I am going to coculture irradiated mouse fibroblast cells with human B cells. I am just wondering if the B cells will attach to the fibroblast cells and if so how I separate them? Thanks.

[miBunny]

B cells don't really attach to the culture flask or to other cells.  You will just rinse them off gently and the fibroblasts should remain adherant.  If you carry over some fibroblasts it won't matter because they will most likely be dead.

[Stephan]

I would add the fibroblasts first, wait for desired confluency and then add the b cells.  It does depend on what you want to do with the cultures afterwards?  I have cocultured breast cancer and natural killer cells before to detect apoptosis.

[TracyDuke]

Thanks to miBunny and Stephan.
Does irradiation just to stop cell proliferation but not kill the cells?

[Stephan]

Sorry TracyDuke, I did not read your question carefully.  Not too sure of the particulars when working with irradiated cells.

[rhombus]

TracyDuke, on Aug 26 2009, 04:56 PM, said:

Hi All, I need to do coculturing in the near future, which I have 0 experience on. I am going to coculture irradiated mouse fibroblast cells with human B cells. I am just wondering if the B cells will attach to the fibroblast cells and if so how I separate them? Thanks.

The easiest way to co-culture is to use cell culture inserts/membranes. These usually fit into multiwell plates. The advantage of using this system is that you can grow your cells on the bottom of the well, then seed your other cells in the insert. The insert/membrane act as a physical barrier to the cells , in that the pore size stops movement of one cell type into the others environment.   Howver any growth factors, cytokines and other cell products COULD permeate the membrane. Perfect if you do not want any cellular cross contamination.
Some people however do studies where they need physical cell-cell interaction. Obviously this system would not suit, and traditionally this type of experiment would be performed in normal TC flasks or dishes. Cross contamination would occur but you could sort these by flfourescently tagging them and then finally sorting them by FACs.

Hope this is useful

Kindest regards

Rhombus