Synchronized turbo apoptosis induced by cold-shock.
In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30-90 min at 37°C more than 80-90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.
Nephrology Research Laboratory, Nijmegen Center for Molecular Life Sciences, Department of Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
J H
JH
Fransen
J W
JW
Dieker
L B
LB
Hilbrands
J H
JH
Berden
J
J
van der Vlag
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis; D001323:Autoantibodies / Q000276:immunology / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D048430:Cell Shape; D048429:Cell Size; D003080:Cold Temperature; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005434:Flow Cytometry; D033162:Incidental Findings; D008180:Lupus Erythematosus, Systemic / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D051379:Mice; D011419:Propidium / Q000032:analysis
D017304:Annexin A5; D001323:Autoantibodies; D011419:Propidium; D020169:Caspases