In this regard, and also relating to other inquiries about Annexin V staining, you might see: Dillon, S.R., M. Mancini, A. Rosen, and M.S. Schlissel. 2000. Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation. J Immunol. 164:1322-32. Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor- mediated signaling events. At 10:53 AM -0400 4/25/00, Carol.W.Johnson@ap.pnu.com wrote: >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 Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 203-688-2748 (fax) mark.shlomchik@yale.edu
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