A flow cytometry assay simultaneously detects independent apoptotic parameters.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
This technique allows us to integrate the analysis and to follow the kinetics of different apoptotic cell changes.
Rasola
A
A
Division of Molecular Oncology, Institute for Cancer Research, University of Torino Medical School, Candiolo, Italy. arasola@ircc.unito.it
Geuna
M
M
D017304:Annexin A5 / Q000378:metabolism; D000968:Antimycin A / Q000494:pharmacology; D017209:Apoptosis; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D048429:Cell Size; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011419:Propidium / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D017304:Annexin A5; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D012235:Rhodamines; D014475:Uncoupling Agents; C401833:tetramethylrhodamine methyl ester; D012402:Rotenone; D011419:Propidium; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000968:Antimycin A; D019311:Staurosporine