I have a drug that when given in vivo to mice increases auto-fluorescence in the APC channel in certain types of immune cells isolated from these mice (methanol fix/perm). What are the possible reasons to explain this?
Ewa
Does your drug accumulate in these channels? Does it fluoresce when exposed to your light source intrinsically?
Hi Michael, thank you for answering. You raised interesting points though I have no idea for now. I have worked with this compound for a long time in in vitro assays (18-h treatment) using the same cells (splenocytes) and combination of antibodies/fluorophores as well and I have never seen it increasing autofluorescence. In vivo I have a subchronic administration (5 days) and i isolate splenocytes also 18 h after the last dose. I do not use methanol fix/perm after in vitro staining however. Could it contribute?
Do you have a fluorescent microscope with the correct laser and filter for APC? If you can see discreet staining it may give you clues. Is it being oxidised in mitochondria and thus acquiring fluorescence that the parent molecule lacks.
Hi Ewa, do you know if your drug is metabolized in vivo to give a product that is autofluorescent?
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