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. 2019 Oct 18;117(10):1805–1819. doi: 10.1016/j.bpj.2019.10.012

Figure 4.

Figure 4

Effect of γ-irradiation on biophysical properties of S. enterica R60. (A) Representative density plots of untreated (1.0 × 108 CFU/mL), γ-irradiated (1000 Gy), or heat-killed (10 min; 95°C) bacteria. Bacteria were analyzed by flow cytometry on a BD FacsCantoII. Size and granularity of bacterial cultures are indicated by the forward scatter (FSC-A) and sideward scatter signal intensities (SSC-A), respectively. (B) The mean ± s.d. of FSC-A and SSC-A values of two (heat-killed) or three independent experiments are shown. (C–E) Flow cytometric measurement of ΔΨ using DiOC2 (3) analyzing differently treated bacteria (untreated, NaN3-treated, and γ-irradiated). (C) FSC-A versus SSC-A was used to gate on bacteria and exclude debris. Additionally, the red channel (RC) versus the width of SSC-W was used to exclude doublets. (D) To calculate the ΔΨ of different treated bacteria, the MFI of the RC and green channel (GC) was measured, and the RC/GC ratio was calculated as described in the Materials and Methods using CCCP treatment for depolarization as individual control for each condition. (E) The ΔΨ of viable bacteria was used as a reference to show its reduction within differently treated bacteria. Measurements were performed using PBS with or without EDTA-treated samples as recommended and described within the BacLight Bacterial Membrane Potential Kit. Calculations and figures regarding the ΔΨ are representative of two independent experiments.