Does anyone have experience with isolation of intestinal macrophages from the lamina propria of the rat intestine or colon? Is there any need for using gradient to extract them or have a better yield? Also, what markers could be used to differentiate or separate the macrophages from DCs in the lamina propria (since gut macrophages are very scarce with the standard macrophage markers and very similar to DCs and plasma cells in the same environment)?
Discontinuous gradient centrifugation will help eliminate debris, undesirable cells, and is relatively simple to use. However, it may not be necessary; you will have to experiment and see what works best for you.
The main challenge you're going to face is in selecting for these cell types; which it sounds like you already are aware of. Macrophages and DCs of the gut express a variety of markers and do not always follow the classic profiles you would expect. Additionally, most of the established literature you're going to find on this issue will be in Mice and Humans which may not translate to Rats. Many subtypes of both cells have been identified all with distinct expression patterns; this complicates things for you.
A place to start may be to negatively select out undesirable cells with CD20/3/8 or similar approach and then use F4/80 to distinguish macrophages from DCs (to my knowledge F4/80 is absent on gut DCs in rodents).
After dissection of the intestinal tissue (collagenase or EDTA-solution in PBS), you can filtrate and wash the suspension in order to remove tissue particles. The next step is to collect the cells using a blood separation medium for mononuclear cells (Ficoll Hypaque, Nycoprep ect). In a third step you can collect MON or DCs by isolation with antibody-coated paramagnetic particles using a permanent magnetic device such as the MACS system (Miltenyi).
I wish you a good success!
Joshua - isn't F4/80 totally absent on rats? I was thinking of using ED1 (CD68), so far it has proven useful in other MF types, though the permeabilisation will diminish the yield of my cells, but as far as my knowledge goes, no one has bothered with ED1 in rat intestinum. I have only found CD11b staining done, and that could mean both MFs and DCs, maybe even some eosinophils and plasma cells...
Josef - do you mean after dissection AND digestion in collagenase? Every protocol I have encountered so far uses colagenase digestion for obtaining lamina propria cells...
Thank you both for your advice
I propose to use Collagenase and stop the digestions with EDTA. SWee pdf file.
Best regards
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