I'm about to make a protocol associating cell sorting with multiparameter flow cytometry (6 colors at least) and microscopy immunofluorescence essay (2 colors) after sorting on sorted cells subsets. I wonder if someone already has done this kind of experiment that has many restriction about the use of fluorochromes. If so, do you have advice concerning a good probe choice for either FACS sorting and microscopy immunofluorescence? I'm using a FACS ARIA III with 4 lasers (UV 355nm,violet 405nm, bleu 488nm, red 633nm). Microscopes are ZEISS apotome 2 and a video microscope with 150W mercury short-arc lamps.
Hi Quentin,
The short version is that you want fluorophores for flow that are either i) identical to what you will use in microscopy, or ii) will not interfere with the three fluorophores you want to use by microscopy. Are you trying to look at cellular targets in BOTH flow and microscopy?
Hi Thomas, Thanks for your interest about this topic.
Actually the staining for flow cytometry must be a surface staining in case of cell culture need for the experiment and the staining for microscopy is intracellular for fixed cell immunochemistry or surface cell staining for time-lapse microscopy.
Hi Quentin, I'm curious about how your experiment worked, since I'm about to be doing the same thing. I've sorted mouse monocytes, and after a quick 15minute stim, proceeded to fix/perm for IF. Were your cells still expressing fluorochromes from the sort?
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