Supravital exposure to propidium iodide identifies apoptosis on adherent cells.
Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.
Zamai
L
L
Institute of Morphological Sciences, University of Urbino, Urbino, Italy. zamai1@biofarm.unibo.it
Canonico
B
B
Luchetti
F
F
Ferri
P
P
Melloni
E
E
Guidotti
L
L
Cappellini
A
A
Cutroneo
G
G
Vitale
M
M
Papa
S
S
D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D002448:Cell Adhesion; D018922:HL-60 Cells; D006801:Humans; D016130:Immunophenotyping; D007202:Indicators and Reagents; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods; D011419:Propidium
D017304:Annexin A5; D007202:Indicators and Reagents; D011419:Propidium