I am studying muscle injury and repair. In one of my models I observe increased necrotic fibers and debris when looking at HE staining of cross-sections. I would like to evaluate phagocytosis and cell debris clearance, but am having trouble finding a good in vivo model. I know of in-vitro models (i.e. culturing macrophages and challenging them with apoptotic or necrotic cells), ex-vivo models (isolate tissue resident macrophages and challenge them with apoptotic or necrotic cells), and even some in-vivo models (inject fluorescently-labeled apoptotic cells into peritoneal cavity measuring clearance OR IV injection and measuring clearance in the spleen).
I have also even read about injecting fluorescently-labeled apoptotic or necrotic thymocytes into injured tissue of live animals and then creating single cell suspensions for clearance by flow. The problem, it doesn't seem very physiological to inject thymocytes from one animal into the muscle of another animal.
Does anyone know of any in vivo methods to evaluate cell/debris clearance or phagocytosis? Preferably by immunohistochemistry.
Dear Steven,
We've been studying phagocytosis in the brain for a while and had similar problems. The only analysis that we found to be sufficiently representative of phagocytosis in vivo appears to be staining with a macrophage marker and a marker of your phagocytic target, i.e. your target cell or structure.Then acquire confocal z-stacks, do 3-dimensional reconstruction and count the number of macrophages containing your target material (see Neher et al., 2013, PNAS). All very tedious and time-consuming unfortunately. Alternatively, you may be able to FAC sort your macrophages, then do Western Blots for proteins of your phagocytic target. Also, markers such as CD68 are being used as a measure of phagocytosis, but are probably not quite as good.
I hope this helps. Good luck!
Jonas
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