We are testing phagocytosis of Borrelia by dendritic cells derivated from murine bone marrow via FACS (the difference in phagocytosis when the cells are stimulated by different substances), but somehow the cells just don't seem to phagocytate. We think it could be caused by the maturation of cell. We're thinking about a different way of derivation than what we use now - anyone has an experience?
Hi, the standard DC differentiation protocol using GMCSF should be used. Around day 4 or 5 you must use the DC (during this period they are immature) for the phagocytosis assay. Please add only Borrelia (if possible fluorescent ?) and any other positive control (DQ Ovalbumin- This works very well and detected in FITC only when the antigen is processing takes place in organelles at low pH. Non processed ova is detected in PE channel. However the color choice is yours as there are different BODIPY conjugates). If you use day 6 or day 7 DC they already have spontaneous maturation (probably due to intrinsic TNFa secretion). Please be sure to do ur experiment on day 4 or maximum at day 5. Coculture DC with the test and controls at 37 deg C for different time points like 0, 5, 15, 30 and 60 mins and within this time points the phagocytosis is already over. I have worked with DQ ova and this works best this way (Atleast this may be used as a positive control for you). Please do not add superantigens like LPS into this set up as this will induce maturation. All the best!
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