Our goal is to use FACS and confocal microscopy to determine if exosomes are entering primary myocytes. We add 5 ul of fluorescently labeled exosomes (concentration 1.0 x 10^8) to a 1.5 ml dish for 24-36 hours. However, we are unable to find a signal. From what we understand, our exosomes and primary cells are fine. Do we need to agitate the culture during those 24-36 hours, add a hormone to the DMEM, etc.? All suggestions are appreciated.
You might want to look at a shorter time point. Once the macrophages pick anything up, the particles are almost always trafficked to the lysosomes. So... by 24 and 36 hours... you exosomes might have already been digested.
If it's just uptake, you don't need to go beyond 4 hours.
Yes I am sort of agree with Shen, 24hrs probably late. these timing are usually picked for the functional assay. If you wanna see only uptaken exosomes 2-4-8hrs (max overnight) should work, you will need to find good exosome concentration. I believe you dont need to shake your cells, and i hope you confirmed your exosome staining worked out.
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