We use GFP as a fate map marker in chimeric mice. We are working on a protocol for a skin digest to analyze the turnover of leukocytes in the skin. The protocol works beautifully on a non-GFP animal, but when GFP is involved, only about 70- 80% of the CD45+ population is GFP+ And the fluorescence is extremely high making compensation extremely difficult. Does anyone have experience in using GFP in the skin of mice. Is this varying expression expected?
Hi Simone,
So if I got this right then the only cells that should be GFP+ are the hematopietic cells and you have a problem with autofluorescence of keratinocytes? You could either gate them out by excluding epcam+ CD45 neg cells or try to get rid of them with a percol gradient. Are the mice BM chimeras? In this case maybe the irradiation was not sufficient and you still get some host-derived CD45+ cells. Also, I believe some fibroblasts/cytes can be CD45+. Best, Sonja
Hi Sonja,
We do work with chimeras but I am having trouble with the GFP control animals. All uncleared cells should be GFP+. Sorry that wasn't clear
Thanks you
What promoter is the GFP under? Is it ROSA? Then this is a bit odd but did you try to gate out the keratinocytes to get rid of the autofluorescence? Also, do you use whole skin or do you separate epidermis and dermis beforehand?
It is under the ubiquiton promoter and it is whole skin, not separated.
Alright, so is it this strain: C57BL/6-Tg(UBC-GFP)30Scha/J? JAX mentions that the GFP expression level in this reporter is twice as high in T cells than in other leukocytes. If you gate on CD45+ in a wt control and a GFP+ sample, are those leukocytes that look negative truly negative or just low for GFP when you compare the histograms of the two samples? Second: are you sure you gated out dying cells and duplets? Usually when cells bite the dust they get leaky and lose GFP signal. Most protocols for skin digestion go in hand with some cell mortality, so what you are looking at may just be dying cells.
I have done my best to gate out dying cells by using a propidium iodide stain. On the histogram there are 3 peaks, one extremely bright and off access, one in the middle and one that I am interpreting as "negative."
What you are sharing could be possible, but in a FACS of the spleen, blood, or even brain tissue we do not see this kind of distribution. I will try gating out the doublets and see if those high expression cells also express T cell markers to be sure.
Thank you for your input!
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