Apoptosis regulation is of fundamental importance in tissue homeostasis and in the pathogenesis of a variety of diseases. Different cytofluorometric methods are used to investigate apoptotic events. We set up a method to simultaneously evaluate mitochondria depolarization, cell morphology changes, and loss of plasma membrane asymmetry and integrity, thus increasing the information and minimizing errors in the analysis of the apoptotic process.

Jurkat T cells were induced to undergo apoptosis with different agents. They were labeled with (1) the mitochondrion‐selective probes tetramethylrhodamine methyl ester (TMRM) or chloromethyl X‐rosamine (CMXRos), which do not accumulate in depolarized mitochondria; (2) Annexin V‐fluorescein isothyocianate (FITC) to detect phosphatidylserine (PS) exposure on the cell surface; and (3) propidium iodide (PI) to assess loss of plasma membrane integrity. Cell morphology changes were studied following variations in light scatter parameters.

This is a fast, reliable, and reproducible technique to detect simultaneously independent apoptotic changes by cytofluorometric inspection. TMRM is more effective than CMXRos in responding to variations in the electrochemical gradient of mitochondria.

This technique allows us to integrate the analysis and to follow the kinetics of different apoptotic cell changes. Cytometry 45:151–157, 2001. © 2001 Wiley‐Liss, Inc.