Figure 6. Heterogeneity of fluorescence within EGFP-expressing population and σ^B activation proportional to the extent of osmotic stress was revealed with FCM. Cells of parent wild-type strain (WT), wild-type-P_lmo2230::egfp (WT-egfp) and ΔsigB-P_lmo2230::egfp (Δσ^B-egfp) derivative strains were grown from a starting OD₆₀₀ = 0.05 up to OD₆₀₀ = 0.6 in BHI (no salt) or BHI supplemented with 0.3 M, 0.6 M or 0.9 M NaCl. FCM was performed using BD Accuri C6 flow cytometer on fixed cells (concentration equivalent to 1 ml of OD₆₀₀ = 0.6, fixed and resuspended in sterile PBS) for three technical replicates of two biological replicates for each strain and each experimental condition. Levels of intrinsic fluorescence (autofluorescence) were determined for wild-type cells without a reporter fusion (WT) and were used to define the gate range of EGFP expressing wild-type-egfp strain population. Mean fluorescence intensities (MFI) for egfp gate and particles located outside the gate (autofluorescence) were calculated by BD CFlow^® software.