Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling

Abstract

Aim: To achieve a better understanding of the pathogenesis of new type gosling viral enteritis virus (NGVEV) and the relationship between NGVEV and host cells.

Methods: The apoptosis of duck embryo fibroblasts (DEF) induced by NGVEV was investigated by fluorescence-activated cell sorter (FACS) and fluorescence microscope after the cells were stained with Annexin V-FITC and propidium iodide (PI).

Results: By staining cells with a combination of fluorescein annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze non-apoptotic cells (Annexin V-FITC negative/PI negative), early apoptotic cells (Annexin V-FITC positive/PI negative), late apoptotic/necrotic cells (Annexin V-FITC positive/PI positive) and dead cells (Annexin V-FITC negative/PI positive) through flow cytometry and fluorescence microscope. The percentage of apoptotic cells increased with the incubation time and reached a maximum at 120 h after infection, while the percentage of non-apoptotic cells decreased.

Conclusion: NGVEV can induce the infected DEF cells to undergo apoptosis and the apoptosis occurs prior to necrosis.

Figure 1

NGVE virus infected DEF cells analyzed by FACS, stained with annexin V-FITC/PI. (A-C) display the results of the cells at 48, 96 and 144 h after NGVE virus infection. The proportion of non-apoptotic cells (c: Annexin V-FITC^-/PI^-), early apoptotic cells (d: Annexin V-FITC⁺/PI^-), late apoptotic/necrotic cells (b: Annexin V-FITC⁺/PI⁺) and dead cells (a: Annexin V-FITC^-/PI⁺).

Figure 2

Flow cytometry of apoptotic DEF cells as assessed by annexin V-FITC fluorescent intensity. DEF cells are mock infected (A) and infected with NGVE virus (B). Cells harvested at 72 h p.i., and subsequently stained with annexin V-FITC/PI. One million cells are analyzed by flow cytometry, data are presented as fluorescent intensity units of annexin V-FITC (abscissa) and number of counts cells (ordinate). The M1 and M2 gates demarcate annexin V-FITC negative populations (non-apoptotic cells) and positive (apoptotic cells) populations.

Figure 3

Apoptotic DEF cells induced by NGVE virus infection stained with Annexin V-FITC/PI and observed under fluorescence microscope. The samples are analyzed for green fluorescence (FITC) and red fluorescence (PI). A: Different labeling patterns of the NGVE virus infected cells: early apoptotic cells, annexin V-FITC positive and PI negative (a and b); necrotic or late apoptotic cells, both annexin V-FITC and PI positive (c-f); dead cells, annexin V-FITC negative and PI positive (g and h); B: 72 h p.i., the early and late apoptotic cells.