Has anyone actually looked at the cells with a fluorescent microscope? We found a similar signal for rat bone marrow cells, but instead of autofluorescence, the cells had speckled surface staining with Annexin V. I agree with Dr Darzynkewicz that microscopy could be of value when an unusual Flow Cytometry signal is present. Carol W Johnson DVM PhD Pharmacia Corp. DARZYNKIEWICZ ZBIGNIEW <DARZYNK@nymc.edu> on 04/22/2000 11:17:20 AM To: cyto-inbox cc: (bcc: Carol W Johnson/USKZO/PNU) Subject: apoptosis and monocytes Maciej Simm wrote: > > One of the people I'm working with right now is studying apoptosis in > periteneal macrophages using a kit with annexin V monoclonal FITC ab > and propidium iodine. > > The cells are autofluorescent. The "unstained" tube has signal in up > to 2nd log. > Monocytes/macrophages may often be positive (by flow cytometry) for several apoptosis markers for the reason that they may have ingested apoptotic bodies detached from genuine apoptotic cells. This is particularly evident e.g. during aggressive chemotherapy of leukemias when many leukemic and perhaps normal lymphocytes die by apoptosis. During ingestion of apoptotic bodies most likely the plasma membrane of the latter fuses with the macrophages' plasma membrane which leads to exposure of phosphatidylserine on the surface and annexin V positivity of macrophages. The ingested apoptotic bodies have fragments of chromatin and thus multiplicity of DNA strand breaks, which makes them also positive in the TUNEL assay (e.g.see Bedner et al., Cytometry, 35: 181-195,1999). I am guessing that such monocytes may also be positive for for other markers e.g. these that detect activated caspases, cleavage of PARP, etc. The only way to identify such "false positive" cells is microscopy or Laser Scanning Cytometry, which allows one to relocate them and examine whether they are loaded with apoptotic bodies. Zbigniew Darzynkiewicz
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