And if so, in what "color"?
I'm running murine bone marrow samples through a flow cytometer and noticed there are some really large cells (about the size of a human cell) that autofluoresce in the FITC (green) channel. I don't think these are macrophages (which are known to autofluoresce in the green channels) because they're much larger.
Any insight would be super helpful. Thanks!
Hello Julia.
Autofluorescence in immune cells is a myth caused by dead or dying cells permeable to free FITC or equivalent present in sec antibody solution.
If you keep your cell fresh and healthy, either live or fixed, it will disappear.
Reduce the concentrations of the pri- and sec-Ab.
Fluorescing cells within the cytoplasm may be evaluated with a dual nuclear staining with propidium iodide (that recognize dying or dead cells).
Wash the cells thoroughly with enough buffer to remove excess free-FITC molecules, too.
Best wishes.
Sang has a great point and I would recommend the propidium iodide staining too! However, we have had a lot of issue with adipocites (i.e. fat cell contamination) within bone marrow and adipose tissue immune cell isolation. They are very large and will autofluoresce in the FITC channel due to the tri-glyceride and high collagen content, the same is found in tissues of high structural components. We managed to remove a lot of this by washing thoroughly and gating out rubbish fragments and I have had no issue with BMDC isolation since! Good luck!
Matt
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