title: Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection.
abstract: Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% +/- 3.56, p < 0.0001, for 24 hours and mean of 37.67% +/- 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.
authors.affiliation: Department of Clinical Analysis, Federal University of Santa Catarina, Florianópolis, SC.
authors.forename: Priscila F R
authors.initials: PF
authors.lastname: Palma
authors.forename: Giovana L
authors.initials: GL
authors.lastname: Baggio
authors.forename: Celso
authors.initials: C
authors.lastname: Spada
authors.forename: Renata D A
authors.initials: RD
authors.lastname: Silva
authors.forename: Silvia Inês A C P
authors.initials: SI
authors.lastname: Ferreira
authors.forename: Aricio
authors.initials: A
authors.lastname: Treitinger
mesh_terms: D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002463:Cell Membrane Permeability; D004996:Ethidium / Q000031:analogs & derivatives / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D015658:HIV Infections / Q000473:pathology*; D006801:Humans; D008214:Lymphocytes / Q000502:physiology*; D011419:Propidium / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017304:Annexin A5; D015415:Biomarkers; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; D011419:Propidium; C018533:ethidium homodimer; D004996:Ethidium