Supravital exposure to propidium iodide identifies apoptotic cells in the absence of nucleosomal DNA fragmentation.
By flow cytometry, we have quantitatively evaluated HL-60, MOLT-4, and P815 apoptotic cells induced with camptothecin, staurosporine, and hyperthermia, respectively. Apoptosis was measured by two different flow cytometry techniques, using propidium iodide (PI) uptake in permeabilized and nonpermeabilized cells. Apoptosis also has been analyzed by electron microscopy and DNA cleavage by in situ nick translation and DNA gel electrophoresis. We demonstrate that supravital exposure to propidium iodide without prior permeabilization identifies apoptotic cells, and clearly distinguishes them from necrotic cells in all the cases examined. This capability is independent of the nucleosomal (180-200 bp) fragmentation of DNA (which does not take place in MOLT-4 cells), which is the basis for detection of apoptosis both as a "ladder" by DNA gel electrophoresis and as a hypodiploid peak by flow cytometry. Therefore, alterations in membrane permeability, on which PI uptake in living cells is based, allow distinction of apoptotic cells from necrotic and living cells independently of the heterogeneous biochemical patterns involved in programmed cell death, which may or may not lead to DNA oligonucleosomal fragmentation.
Istituto di Anatomia Umana, Università di Bologna, Italy.
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L
Zamai
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E
Falcieri
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G
Marhefka
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M
Vitale
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D004396:Coloring Agents / Q000494:pharmacology*; D004249:DNA Damage / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D009707:Nucleosomes; D011419:Propidium / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004396:Coloring Agents; D009707:Nucleosomes; D011419:Propidium