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. 2015 Sep 25;10(9):e0138777. doi: 10.1371/journal.pone.0138777

Fig 1. Representative cytograms for simultaneous flow cytometric assessment of subtle membrane changes, viability and reactive oxygen species (ROS).

Fig 1

Samples were stained with Hoechst 33342, Yo-Pro-1, ethidium homodimer (Eth) and CellROX Deep Red, as described in the ‘Materials and methods’ section. Hierarchical gating was applied to exclude debris from the analysis and simultaneously measure viability, apoptosis, necrosis and ROS. (A) Hoechst 33342 fluorescence was detected using the V1 channel (Ex 405 bandpass filter 450/50 nm), and a gate was applied to positive (DNA-containing particles) events to gate out debris. The gated region was analysed. (B) Yo-Pro-1 was detected using the B1 channel (Ex 488 bandpass Filter 525/50 nm) and Hoechst 33342 fluorescence was detected using the V1 channel (Ex 405 bandpass filter 450/50 nm) (C,) Yo-Pro-1 was detected using the B1 channel (Ex 488 bandpass Filter 525/50 nm) and Eth was detected using the B3 channel (Ex 488 bandpass filter 655–730 nm) and (D) CellROX Deep Red was detected using the R1 channel (Ex 635 nm bandpass filter 655–730 nm). Positive controls for oxidation and compromised membranes are presented obtained as describe in material and methods.