Apoptosis assays with lymphoma cell lines: problems and pitfalls.
Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death.
Mattes
M J
MJ
Garden State Cancer Center at the Center for Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville, NJ 07109, USA. mjmattes@gscancer.org
D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000502:physiology*; D001562:Benzimidazoles / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D002449:Cell Aggregation / Q000502:physiology; D045744:Cell Line, Tumor; D024161:Collagen Type XI / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008223:Lymphoma / Q000378:metabolism / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011419:Propidium / Q000737:chemistry
D017304:Annexin A5; D001562:Benzimidazoles; C499319:COL11A2 protein, human; D002232:Carbocyanines; D024161:Collagen Type XI; D004273:DNA, Neoplasm; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; D011065:Poly(ADP-ribose) Polymerases