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Synchronized turbo apoptosis induced by cold-shock.
J H Fransen1, J W Dieker, L B Hilbrands
1Nephrology Research Laboratory, Nijmegen Center for Molecular Life Sciences, Department of Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
Apoptosis : an International Journal on Programmed Cell Death
|October 26, 2010
Summary
We developed a rapid cold-shock method to generate synchronized apoptotic cells for studying systemic lupus erythematosus (SLE). This technique efficiently produces homogeneous early and late apoptotic cells and blebs for research.
Area of Science:
- Cell Biology
- Immunology
- Pathogenesis of Autoimmune Diseases
Background:
- Apoptosis plays a role in systemic lupus erythematosus (SLE) pathogenesis.
- Understanding the effects of apoptotic cells on antigen-presenting cells is crucial for SLE research.
- Generating synchronized, homogeneous populations of early and late apoptotic cells in vitro is challenging.
Purpose of the Study:
- To develop a rapid, reproducible method for generating synchronized homogeneous populations of early and late apoptotic cells and blebs.
- To investigate the characteristics of cold-shock-induced apoptosis.
- To assess the utility of these apoptotic cells in SLE-related research.
Main Methods:
- Granulocytic 32Dcl3 cells were incubated on ice for 2 hours, then rewarmed at 37°C.
- Apoptosis was induced via cold-shock followed by rewarming.
- Apoptotic cells and blebs were characterized using Annexin V and propidium iodide staining.
- Caspase activity and chromatin cleavage were assessed.
- Binding of SLE-derived anti-chromatin autoantibodies to blebs was evaluated.
Main Results:
- Cold-shock followed by rewarming rapidly induced synchronized apoptosis in 32Dcl3 cells.
- 80-90% early apoptotic cells (Annexin V+/PI-) were observed after 30-90 minutes at 37°C.
- 98% late apoptotic/necrotic cells (Annexin V+/PI+) were observed after 24 hours.
- Apoptotic blebs formed within 20 minutes at 37°C.
- Inter-nucleosomal chromatin cleavage and caspase activation were confirmed.
- Caspase inhibition blocked apoptosis.
- SLE-derived anti-chromatin autoantibodies showed high affinity for cold-shock-induced blebs.
Conclusions:
- Cold-shock is an effective, non-toxic method for rapidly generating synchronized, homogeneous apoptotic cell populations.
- This method provides a valuable tool for studying apoptosis in various contexts, including SLE pathogenesis.
- The generated apoptotic blebs are recognized by SLE-specific autoantibodies, suggesting relevance for disease mechanisms.
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