Mark,
Thanks for the note. It would make sense that this is the problem, since the
fluorescent pattern was more like that which I have observed with other
lymphocyte markers and was different from that observed with usual apoptosis.
We have been wondering how to kill the 'nonspecific' binding in the bone marrow.
Your reference to the paper suggests the problem is reagent cross-reactivity and
not non-specific binding. With up to 60% of rodent bone marrow being composed
of B-lymphocytes, it does not seem that Annexin V will be a good technique to
measure apoptosis in rat bone marrow.
Do you (or anyone else) have any other suggestions as to how we can measure
apoptosis or cell death in the bone marrow? We are working on some drugs with
some unusual toxicologic effects on B-cells and other cell types in the bone
marrow of rats.
Regards,
Carol W. Johnson DVM PhD
Pharmacia
Mark Shlomchik <mark.shlomchik@yale.edu> on 05/02/2000 05:58:57 PM
To: cyto-inbox
<cytometry@flowcyt.cyto.purdue.edu>
cc:
Subject: Re: apoptosis and monocytes
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
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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