Fig 1.
Calcein labels mammalian viable cells and dead but not viable Leishmania cells.
(A-B) Raw histograms from flow cytometer analysis of viable THP1 cells (A) or viable L. major cells (B) stained or not stained with calcein-AM. The percentage of calcein-positive cells is given in brackets in the legend. All viable THP1 cells appeared stained with calcein-AM, while viable L. major cells were not stained. The same result has been obtained in at least four independent experiments. (C) Raw histograms from flow cytometer analysis of viable L. major cells (WT) and of apoptotic cells stained with calcein-AM. Cell apoptosis was induced for 24h either with 50μM of curcumin, 600μM of H2O2, 40μM of miltefosine, or 100μM of pentamidine. (D) Mean percentage (±SD) of calcein-positive cells in viable WT cells and after the addition of four pro-apoptotic drugs for 24h. The number of experiments performed (n) is given for each condition. Unpaired t-test, ** p<0.01 and *** p<0.001. (E) Microscopical observation of WT L. major cells (upper panels) and L. major cells treated with 40μM of miltefosine for 24h (lower panels) and labeled with calcein-AM. Calcein gathered in some dead cells in punctuated cytoplasmic structures (arrow) (bar = 5μm).
Fig 2.
Calcein/PI labeling makes it possible to distinguish early apoptosis from late apoptosis and necrosis.
(A-B) Quantification of calcein and PI staining in WT cells and in cells incubated with 40μM of miltefosine for 8h, 24h and 48h: raw data for one experiment representative of at least four experiments (A) and mean percentage (±SD) of cells in early apoptosis (calcein+/PI-), late apoptosis (calcein+/PI+) and necrosis (calcein-/PI+). Unpaired t-test, * p<0.05, ** p<0.01 and *** p<0.001. (C-D) Quantification of calcein and PI staining in WT cells and in cells incubated with increasing concentrations of miltefosine: 20μM, 40μM or 100μM for 24h: raw data for one experiment representative of at least six experiments (C) and mean percentage of cells ±SD (D). Unpaired t-test, * p<0.05, ** p<0.01 and *** p<0.001.
Fig 3.
Calcein+/PI- is a very early apoptotic feature of Leishmania cells.
(A) Percentage of cells in early apoptosis or in late apoptosis/necrosis, according to a calcein/PI labeling or to a TUNEL assay: mean ±SD. Cells were considered to be in early apoptosis when they were calcein+/PI- according to the calcein/PI labeling and TUNEL-positive with a non-degraded nucleus according to the TUNEL assay. For late apoptosis/necrosis, PI-positive cells (calcein/PI labeling) and cells with a degraded or lost nucleus (TUNEL assay) were considered. Significant differences appeared between both techniques concerning the percentage of early apoptotic cells, whereas the percentages of late apoptotic/necrotic cells were the same with the two assays, according to three independent experiments. Unpaired t-test, * p<0.05 and ** p<0.01. (B) Correlation between the percentage of PI-positive cells and of cells without a nucleus. (C) Percentage of calcein-positive cells after incubation of the cells for 20min, 40min or 120min with 40μM of miltefosine: mean ±SD. Unpaired t-test, * p<0.05 and ** p<0.01.
Fig 4.
Model: calcein/PI labeling in Leishmania.
In viable cells, the membrane properties prevent calcein and PI from entering the cells which appear calcein-/PI-. During early apoptosis, very early membrane modifications induce a high plasma membrane permeability, inducing calcein entry. Membrane integrity maintenance prevents PI from entering the cells. As a consequence, cells in early apoptosis appear calcein+/PI-. During late apoptosis, membrane degradation creates pores in the plasma membrane allowing PI to enter the cells which are calcein+/PI+. Finally, in vitro, in the absence of phagocytosis, cells enter necrosis characterized by membrane loss, so these last cells appear calcein-/PI+.