I want to establish peritoneal macrophage migration assay by 8.0μm boyden chamber.Is it possible to compare the cell migration ability in different genotype of cells,for example WT cells and KO cells in response to chemokines. In this case,it would be 3 parameters changed. First,I think the cell number seeding in the chamber is not totally equal even if determining the cell number by cell counter.Second, cell genotype is different.Third,treatment is different (within or without chemokine in below well)
I think your plan sounds good, it is all possible. I did use this assay for different reason in the paper enclosed. That might give you a small 'go ahead" support and thinking,
You can use KO cells for transwell migration assay. Make sure to minimize counting errors of cells when add into chamber. You can increase number of replicate to minimize error. To compare the results need to add same amount of cells to transwell chambers.
I think that you have several other parameters that appear to be outside your control here. In the first place your macrophage population is not homogeneous, rather it is a mixture of maturities and state of activation. Heterogeneity will be even greater after manipulation/KO.
That said I still think that you may be able to get some useful data from your assay.
Please use a manual count, not a machine, to ensure your cell numbers are accurate!
Remember that your cells are populations! They are not clonal and not all equal!
The key to the Boyden chamber assay is timing. If you marginalise the assay (e.g. shorter times, serum-free, hypoxia) you will maximise your signal to noise ratio and hopefully observe a measurable difference between your cell populations.
How will you verify you have KO'd transcription in your KO cells?
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