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. Key-Words: Apoptosis, flow cytometry, annexin V, propidium iodide, calcein-AM, ethidium homodimer. Abbreviations: Calcein-AM, calcein acetoxymethyl ester; EthD-1, ethidium homodimer; FITC, fluorescein isothiocyanate.

Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells

“…Cells were visualized by the live-cell dye Calcein violet AM (blue), early-apoptotic and dead cells were visualized by Annexin V Alexa Fluor ® 488 (green) and propidium-iodide (red) staining. Annexin V was applied to detect phosphatidyl-serine exposure of apoptotic cells and PI to detect late apoptotic and necrotic death (Bratosin et al 2005; Palma et al 2008). Between days 4 and 7, gradual morphological changes were noticed with the outspreading of fibroblast-like cells in the monolayer cultures (Fig.…”

Change of Morphological and Functional Characteristics of Retinal Pigment Epithelium Cells during Cultivation of Retinal Pigment Epithelium-Choroid Perfusion Tissue Culture

“…Therefore, it can be utilized to distinguish live and dead cells through the cytoplasm green fluorescence intensity. Furthermore, calcein-AM provides morphological evidence of apoptotic changes such as chromatin condensation and segregation in blebs, together with functional information about plasma membrane integrity and intracellular esterase activity [8,9] .…”

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

“…To assess the consequence of G5-NH 2 induced Ψ MITO depolarization we measured the viability of astrocytes and neurons by labeling the live cells with calcein after 30 min exposure to G5-NH 2 . The SR101 positive astroglial cells showed robust calcein fluorescence in the stratum radiatum after 30 min of G5-NH 2 application indicating the presence of functional, viable astroglial cells [28] (Figure 6A Top, n = 3 slices), although viability of astrocytes in the stratum lucidum region may also be compromised. Contrary, in accordance with our previous observations [13], a large proportion of hippocampal pyramidal neurons lost their viability after 30 min application of G5-NH 2 despite the survival of astrocytes in the same region (Figure 6 Middle and Bottom ).…”

“…The intracellular esterase activity could be used as a probe of viability and plasma membrane competence and as an indicator of the cellular functionality [28]. Calcein-AM is a membrane-permeable non-fluorescent molecule that enters intact living cells, then it is cleaved by endogenous esterases to produce the highly fluorescent, membrane impermeable molecule, calcein.…”