Using laser scanning cytometry to analyze apoptosis

Michael J. Lander

Research that helps scientists to efficiently assess the response of human cells to drugs could aid in drug development and in ongoing treatment of patients. Studies that deal with apoptosis, in particular, might allow clinicians to better treat cancer patients.

Shazib Pervaiz and colleagues from the Yong Loo Lin School of Medicine, National University of Singapore have used laser scanning cytometry in a 96-well format to analyze drug-induced apoptosis in tumor cells. The technology, which measures multicolor fluorescence and light scatter, has enabled them to gain more insight into apoptosis by imaging simultaneously morphological and biochemical events in the same cell population.

In their study, published in the Jan. 2 online edition of Cytometry Part A, the scientists employed a CompuCyte laser scanning cytometer with 450-nm, 488-nm argon and 633-nm HeNe lasers and four filter cube sets. They induced an apoptotic response in experimental cells with 2-mercaptopyridin-N-oxide hydrate sodium salt.

The researchers first differentiated and counted cell clusters and segmented single cells with a cell-permeable double-stranded DNA nuclear dye. They created histograms of the DNA profiles from this data and discovered G1 cell cycle arrest in many cells. To assess strand breaks and nuclear DNA content, they created a density plot showing DNA content versus staining for histone H2AX phosphorylation at Ser139, which illuminated live, apoptotic and dead bodies. Further imaging with the cytometer’s confocal platform identified pronounced hollowing in treated cells’ nuclei. These characteristic changes indicated that the agent had caused apoptosis in many cells.

The investigators recorded changes in morphology and cell membrane permeability as well. Transformations of the former type included cell rounding and detachment. A trio of red, green and blue dyes for nucleic acids showed that cells exposed to the chemical took up dye at a higher rate, indicating early apoptosis.

Finally, the researchers noticed that mitochondria became rounder and smaller in experimental cells. Evaluation with a mitochondrial potential assay demonstrated that, as the compound’s concentration increased, mitochondria took up a cationic probe more readily — an indication of membrane potential loss. The team’s simultaneous accurate detection of these various phenomena associated with programmed cell death demonstrated the efficiency of laser scanning cytometry compared with other techniques in this application.

In future investigations, the team intends to use the system to monitor protease activity with Förster resonance energy transfer and to evaluate cytosolic pH and the formation of reactive oxygen species. Through such work, they hope to gauge the potential of the system for analyzing other diseases.