Hi there,
I am tending to do triple culture (Epithelial cells, fibroblasts and macrophages in order to test cells migration, invasion and proliferation in 96 well plate. Can anyone tells me how many cells (each) do I need to see ? I am thinking to culture equal number of cells but I am not sure if I am correct or not. Also has anyone done it before please ?
Thanks in advance
Hi Ahmed,
Could you please explain in more details what methods you are going to use to measure cell migration/ invasion and proliferation? 96-well scatch? Transwell chambers? BrdU / MTT assay? You may need to use different cell numbers for different assays and cell types.
The best would be to do a cell titration (50000 , 100000 , ...) separately for each type.
Best
Hi Ahmed,
Indeed, cell number will strongly depends on assay you going to use for estimation of cell migration.
In general it will also depend on your goal. If you going to imitate some structures you will probably need to create fibroblast monolayer doped with macrophages first and later cover it with epithelial cells. I'm not sure if you need to prevent fibroblast growing after monolayer formation or not. Reading on feeder layer protocols could help a lot to develop appropriate experimental setup.
Similar approach was used to form in vivo-like structures using bronchial epithelial cell based multilayer structures for evaluation of radiation effects.
Human keratinocites as well could form skin-like structures.
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General rule to use starting sell number giving you those close to normal tissue ratio for different cell types. Certain numbers will depend of surface area you going to use in your experiment.
Hope it will help to find right direction.
Daniil
Hi Vida
Thank you for the reply, regarding to your questions, yes I will use Live cell imaging from plates, Live cell imaging, 96 well plates, Fluorescent Microscope. This system has been widely used by many researchers at Leeds university to measure cell proliferation, migration and invasion. Yes I will be able to use only 96 well plates ( scratch assay for cells migration). Have you ever used this method ?
Hi Daniil
Thank you very much for your reply. Yes I agree with you that cells number is very important, and I have to be really cautious which picking a cell number especially when using double or triple culture technique. However, I think I would go with various numbers of cell number in order to avoid biasing and hopefully I will find something interesting.
Cheers
Ahmed
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