Hi,
I want to set up an experiment to analyze ability of THP-1 derived macrophages to kill MC7-7 cells. But both of them are adherent cells, which makes it difficult to culture them in one plate. I thought to use Transwell membrane for coculture. I am not sure that macrophages can contact and kill MCF-7 cells in Transwell membrane plates. Do these cells interact via Transwell membrane?
Thank you
THP-1 cells would probably migrate through the membrane pores, so I wouldnt do that. In theory, it shouldnt be a problem at all. What kind of read-out are you using to assess killing? If it is a Chromium-release assay, you need the contact anyway, and based on how the assay work, you need a direct contact. If you use LDH release, the problem will be more to have appropriate negative controls, for FACS based analysis, the same as for the Cr51-release assay accounts.
Yes. You can co-culture them. Use the Sigma PET coated 0.4 uM pore sized membranes. Cells can also be cultured using matrigels 3D gel system as an alternative. Do your cells secrete the chemokines for the migrations towards each other or they will need direct physical contact only?
Hello,
It is possible to co-culture those cell lines but, the question is: how can you recognise MC7-7 from THP-1? It is fine if you are planning to use the co-culture lysate. the best thing to do is to seed the non migrating cells first, then tag the migrating cells with specific dye and add it to the culture. that is the way to study the migrating cells behaviour.
Best wishes
not sure why you do not want to co-culture them on adherent plates. I would put fluorescent marker in one or bot (e.g. dsRed or EGFP) and then you can follow in real time what is going on. I would do it both ways as reviewers will want to make sure the fluorescent proteins are not affecting the results. Thus for MCF-7 add nothing, red and green and do the same for THP-1. I suggest getting lentivral vectors (easily available from most sources)
You can indirectly (separate) co-culture by using transwell of pore size 0.4 micron but for migration studies you can use the transwell of pore size of 3 micron or larger. You can also co-culture directly in the same plate by marking them with different color fluorescent markers.
You can co-culture and quantify both the cells. For that you have to arrest the mitosis of MCF-7 (mitomycin will help) and seed a specified number of cells. These cells stop division but can survive for 4-5 days. To a layer of cell cycle arrested cells you have to seed specified number of ThP1 cells. For quantification MTT assay with appropriate controls can be followed.
Maybe you want to check the following review, which summarizes current co-culture methods and their advantages/disadvantages.
Methods for co-culturing tumour and endothelial cells:
systems and their applications
Mallory van Moorst and Crispin R. Dass
DOI
10.1111/j.2042-7158.2011.01352.x
Culturing two different cells that are growing as adherent should not be a problem in your culture. The important issue is how you are going to measure the killing effects of the macrophages? If it is important to distinguish the two cells morphologically, then you have to tag one of them with an appropriate florescent markers.
Good luck
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