Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip
Introduction: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required.
Methods: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-α in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer.
Results: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used.
Conclusions: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.
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- 1.Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. J Immunol Methods 2000; 243: 167–190.Google Scholar
- 2.Vermes I, Haanen C. Apoptosis and programmed cell death in health and disease. Adv Clin Chem 1994; 31: 177–246.Google Scholar
- 3.Reyes D, Iossifidis D, Auroux P, Manz A. Micro total analysis systems. 1. Introduction, theory, and technology. Anal Chem 2002; 74: 2623–2636.Google Scholar
- 4.Aroux P, Iossifidis D, Reyes D, Manz A. Micro total analysis systems. 2. Analytical standard operations and applications. 2002; 74: 2637–2652.Google Scholar
- 5.Van den Berg A, Lammerink T. Micro total analysis systems: Microfluidic aspects, integration concept and applications. Top Curr Chem 1998; 194: 21–49.Google Scholar
- 6.Andersson H, van den Berg A. Microfluidic devices for cellomics: a review. Sensors Actuators B 2003; 92: 315–325.Google Scholar
- 7.Andersson H, van den Berg A. Microtechnologies and nanotechnologies for single cell analysis. Curr Opin Biotechnol 2004; 15: 44–49.Google Scholar
- 8.Guijt RM, Baltussen E, van der Steen G, et al. New approaches for fabrication of microfluidic capillary electrophoresis devices with on-chip conductivity detection. Electrophoresis 2001; 22: 235–241.Google Scholar
- 9.Blom MT, Chmela E, Oosterbroek RE, Tijssen R, van den Berg A. On-chip hydrodynamic chromatography separation and detection of nanoparticles and biomolecules. Anal Chem 2003; 75: 6761–6768.Google Scholar
- 10.Vrouwe EX, Luttge R, van den Berg A. Direct measurement of lithium in whole blood using microchip capillary electrophoresis with integrated conductivity detection. Electrophoresis. Accepted for publication 2004; 24.Google Scholar
- 11.Dellinger M, Geze M, Santus R, et al. Imaging of cells by autofluorescence: A new tool in the probing of biopharmaceutical effects at the intracellular level. Biotechnol Appl Biochem 1998; 28: 25–32.Google Scholar
- 12.Knight AW, Billinton N. Distinguishing GFP from cellular autofluorescence. Bioph Int September/October 2001.Google Scholar
- 13.Aubin JE. Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem 1979; 27: 36–43.Google Scholar
- 14.Benson RC, Meyer A, Zaruba ME, McKhann GM. Cellular autofluorescence-Is it due to flavins? J Histochem Cytochem 1979; 27: 44–48.Google Scholar
- 15.DaCosta RS, Andersson H, Wilson BC. Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy. Photochem Photobiol 2003; 78: 384–392.Google Scholar
- 16.Andersson H, Baechi T, Hoechl M, Richter C. Autofluorescence of living cells. J Microsc 1998; 191: 1–7.Google Scholar
- 17.Petty HR, Worth RG, Kindzelskii AL. Imaging sustained dissipative patterns in the metabolism of individual living cells. Phys Rev Lett 2000; 84: 2754–2757.Google Scholar
- 18.Brock R, Hink MA, Jovin TM. Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys J 1998; 75: 2547–2557.Google Scholar
- 19.Wallach D, Varfolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP. Tumor necrosis factor receptor and Fas signalling mechanisms. Ann Rev Immunol 1999; 17: 331–367.Google Scholar
- 20.Lui LF, Desai SD, Li TK, Mao Y, Sun M, Sim SP. Mechanism of action of camptothecin. Ann N Y Acad Sci 2000; 922: 1–10.Google Scholar
- 21.Overbeeke R, Yildrim M, Reutelingsperger CPM, Haanen C, Vermes I. Sequential occurrence of mitochondrial and plasma membrane alterations, fluctuations in cellular Ca2+ and pH during initial and later phases of cell death. Apoptosis. 1999; 4: 455–460.Google Scholar
- 22.Parone PA, James D, Martinou JC. Mitochondria: Regulating the inevitable. Biochimie 2002; 84: 105–111.Google Scholar
- 23.Nieminen A. Apoptosis and necrosis in health and disease: Role of mitochondria. Int Rev Cytol 2003; 224: 29–55.Google Scholar
- 24.Lorenzi M, Cagliero E, Toledo S. Glucose toxicity for human endothelial cells in culture. Diabetes 1985; 34: 621–627.Google Scholar
- 25.Baumgartner-Parzer SM, Wagner L, Pettermann M, Grillari J, Gessi A, Waldhäusl W. High-glucose-triggered apoptosis in cultured endothelial cells. Diabetes 1995; 44: 1323–1327.Google Scholar
- 26.Carpentier Y, Mayer P, Bobichon H, Desoize B. Cofactors in in vitro induction of apoptosis in HL60 cells by all-trans retinoic acid (ATRA). Biochem Pharmacol 1998; 15: 177–184.Google Scholar
- 27.Emmelkamp J, Wolbers F, Andersson H, et al. Autofluorescence of single living cells for label-free cell sorting in microfluidic systems. Submitted.Google Scholar