A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell
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Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10−15 g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.
KeywordsApoptosis Bioluminescence Caspase 3/7 Single-cell analysis
Tetrapeptide sequence Asp-Glu-Val-Asp
The research was supported by the Grant Agency of the Czech Republic, projects 14-28254s (at the IAC ASCR, v.v.i.), P304/11/1418 (at the IAPG ASCR, v.v.i.), and P502/12/1285 (at the UVPS). The research runs also under the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101). The authors wish to thank Mgr. Jan Křivánek for the kind gift of human stem cells.
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