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. 2020 Feb 26;11:291. doi: 10.3389/fmicb.2020.00291

FIGURE 2.

FIGURE 2

Commensals aid maintenance of monolayer integrity during pathogen colonization. HaCat monolayers were stained with CellTracker deep red prior to incubation with commensals and/or S. aureus for 40 h. Where present, P. aeruginosa was included for the final 20 h of the incubation. Bacterial inoculation ratios were as described in Figure 1, and images taken at 63 × magnification. Scale bar indicates 20 μm. HaCat cells were inoculated with (A) no bacteria; (B) commensals S. epidermidis and M. luteus; (C) P. aeruginosa; (D) P. aeruginosa and commensals; (E) S. aureus; (F) S. aureus and commensals; (G) P. aeruginosa and S. aureus; (H) HaCat P. aeruginosa and S. aureus and commensals. HaCat cells are shown as a representative single plane. (I) Quantification of HaCat monolayer integrity by measuring the total fluorescence of the z-stack HaCat cells using ImageJ (8–10 Z-stack images analyzed per condition, two experiments), and plotted as percentage of the HaCat only control. SE: S. epidermidis; ML: M. luteus; SA: S. aureus; PA: P. aeruginosa. ∗p-value < 0.05.