An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells

Abstract

Current methodologies to measure apoptotic and necrotic cell death using flow cytometry do not adequately differentiate between the two. Here, we describe a flow cytometry methodology adapted to airway epithelial cells (AEC) to sufficiently differentiate apoptotic and necrotic AEC. Specifically, cell lines and primary AEC (n = 12) were permeabilized or infected with rhinovirus 1b (RV1b) over 48 h. Cell death was then measured via annexin V/propidium iodide (A5/PI) or annexin V/TO-PRO-3 (A5/TP3) staining using a novel flow cytometry and gating methodology adapted to AEC. We show that A5/PI staining could not sufficiently differentiate between types of cell death following RV1b infection of primary AEC. However, A5/TP3 staining was able to distinguish six cell death populations (viable, necrotic, debris, A5⁺ apoptotic, A5^- apoptotic, apoptotic bodies) after permeabilization or infection with RV1b, with phenotypic differences were observed in apoptotic populations. Collectively, using a staining and gating strategy never adapted to AEC, A5/TP3 could accurately differentiate and quantify viable, necrotic, and apoptotic AEC following RV1b infection.

Keywords: airway epithelium; cell death; flow cytometry; rhinovirus.

Figure 1.

A5/TP3 gating and data analysis for differentiation of a sample into six populations. A. Data was sorted into TP3⁺ events and other events. B. The A5⁺/TP3⁺ events were sorted by FSC into necrotic events (FSC⁺). C. The A5^–/TP3^– events were then separated by SSC and A5 into A5^–/SSC⁺ and other events. D. The A5^–/SSC⁺ events were then sorted into viable (FSC⁺/TP3^–) and A5^– apoptotic events (TP3⁺). E. The “other events” were then separated into A5⁺ and A5^–, with the A5^– then sorted by FSC into cellular debris (FSC^–) (F). G. The A5⁺ events were then sorted by FSC into A5⁺ apoptotic events (FSC⁺) and apoptotic bodies (FSC^–).

Figure 2.

A5/PI cannot differentiate types of cell death in primary AEC but A5/TP3 is effective to differentiate viable, apoptotic, and necrotic AEC lines. Primary AEC (n = 4) were infected with RV1b for 48 h, collected and stained with A5/PI. RV1b infection decreased viable events (A), had no effect on necrotic events (B), had no effect on apoptotic events (C), and increased secondary necrotic events (D). AEC lines (n = 7) were collected, fixed with paraformaldehyde, and analyzed via flow cytometry. After fixation with paraformaldehyde, viable events were reduced compared to control, necrotic events were increased compared to control, debris was reduced compared to control, A5⁺ apoptotic events were increased compared to control, and no changes in apoptotic bodies or A5^– apoptotic events were observed compared to controls (E). When grouped into “viable”, “necrotic”, and “apoptotic” events, viable events were reduced compared to control (F), necrotic events were increased compared to control (G), and apoptotic events were increased compared to control (H). *P < 0.05.

Figure 3.

A5/TP3 staining in primary AEC results in measurable changes in viable, necrotic, and apoptotic events via flow cytometry. Primary AEC were collected, fixed with paraformaldehyde (n = 4) or infected with RV1b (n = 12) over 48 h, stained with A5/TP3, and analyzed via flow cytometry. After fixation with paraformaldehyde, viable events were reduced (A), necrotic events were increased (B), and apoptotic events were increased (C). RV1b infection: decreased viable after infection for 24 h and 48 h, with lower viable events at 48 h compared to 24 h in both control and RV1b (D), increased necrotic events after RV1b infection for 24 h and 48 h, with increased necrotic events at 48 h compared to 24 h (E), significantly increased debris following RV1b infection for 24 h (F) no change in A5⁺ apoptotic events after RV1b infection over 48 h (G), significantly increased apoptotic bodies following RV1b infection for 24 and 48 h, with higher apoptotic bodies after 48 h of infection compared to 24 h (H), and significantly decreased A5^– apoptotic events following RV1b infection for 24 h and 48 h (I). *P < 0.05.

Figure 4.

A5/TP3 staining in primary AEC results in measurable phenotypic differences in apoptotic populations. Non-CF (n = 6) and CF (n = 6) primary AEC were infected with RV1b over 48 h, stained with A5/TP3, and analysed via flow cytometry. RV1b infection: had no significant effect on A5⁺ apoptotic events after 24 h (A) and 48 h (B), significant decreased A5^– apoptotic events after 24 h in both non-CF and CF AEC, with significantly lower events in CF (C), significantly decreased A5^– apoptotic events in CF AEC after 48 h, with significantly lower events in CF compared to non-CF AEC (D), significantly increased apoptotic bodies in non-CF AEC after 24 h (E), and significantly increased apoptotic bodies in non-CF and CF AEC following 48 h of RV1b infection (F). *P < 0.05.