Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis.
(1999) Cell Calcium — Vol. 25, n° 1, p. 19-28 (1999)
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- Abstract
- Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.
- Affiliations
Baltimore John Hopkins Oncology Center
Citations
Tombal, B., Denmeade, S. R., & Isaacs, J. T. (1999). Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis. Cell Calcium, 25(1), 19-28. https://doi.org/10.1054/ceca.1998.0005 (Original work published 1999)