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. 2018 Apr 4;13(4):e0194855. doi: 10.1371/journal.pone.0194855

Fig 4. Monocyte fraction persistence.

Fig 4

(A—E) For analysis, freshly isolated monocytes were incubated in pooled supernatant from MDM stimulated with corneal tissue; monocytes were identified using flow cytometry (depicted for six (A and B) and 72 hours (D and E) of incubation). The monocyte fraction was determined based on the percentage of gated cells shown in flow cytometry plots. FACS was performed after six, 24, 48 and 72 hours of incubation (Fig 4C). Counting beads were used to ensure consistent cell counts in all samples. The presence (blue) or absence (red) of corneal endothelium (CEC) during the initial MDM stimulation is indicated. In the absence of CEC twice as many monocytes can be found after six hours of incubation. Gated monocytes (as in A, B, D, and E) were further sub-differentiated into classical (CD14++CD16−), intermediate (CD14++CD16+), and non-classical (CD14+CD16++) monocytes (Fig 4F). Monocytes incubated with supernatant from MDM stimulated with endothelium showed a statistically significant increase in intermediate monocytes (Fig 4F and G; * p<0.05).