Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer.
BACKGROUND
Here we demonstrate the ability of the ImageStream 100 Multispectral Imaging Cytometer to discriminate between live, necrotic, and early and late apoptotic cells, using unique combinations of photometric and morphometric features.


METHODS
Live, necrotic, and early and late apoptotic cells were prepared and analyzed by immunofluorescence microscopy, conventional flow cytometry, and imaging flow cytometry, both as single populations and as a heterogeneous mixture of cells.


RESULTS
Live (annexin V(-), 7-AAD(-)) and early apoptotic (annexin V(+), 7-AAD(-)) cells were readily identifiable using either conventional or ImageStream based flow cytometric methods. However, inspection of multispectral images of cells demonstrated that the annexin V(+), 7-AAD(+) population contained both necrotic and late-stage apoptotic cells. Although these cells could not be distinguished using conventional flow cytometric techniques, they were separable using unique combinations of photometric and morphometric measures available using ImageStream technologies.


CONCLUSIONS
Using multispectral imagery, morphologically distinct cell populations can be distinguished using features not available with conventional flow cytometers. In particular, the ability to couple morphometric with photometric measures makes it possible to distinguish live cells from cells in the early phases of apoptosis, as well as late apoptotic cells from necrotic cells.
George
Thaddeus C
TC
Amnis Corporation, Seattle, Washington 98121, USA. t.george@Amnis.com
Basiji
David A
DA
Hall
Brian E
BE
Lynch
David H
DH
Ortyn
William E
WE
Perry
David J
DJ
Seo
Michael J
MJ
Zimmerman
Cathleen A
CA
Morrissey
Philip J
PJ
D016923:Cell Death / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005455:Fluorescent Antibody Technique; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods