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. 2015 Jan 26;5:8008. doi: 10.1038/srep08008

Figure 7. NET microbicidal activity on Cryptococcus neoformans.

Figure 7

B3501 wild-type yeasts were incubated overnight in the absence or presence NET-enriched supernatants, and stained with calcofluor or the Live/Dead probe FUN®1 (live yeasts metabolize green FUN1 to a red fluorescent dye; dead yeasts remained green). (A)B3501 + control supernatant stained with calcofluor; (B, C) B3501 + control supernatant labeled with FUN1;(D) Overlay of B and C images depicting live yeasts. (E) B3501 + NET-enriched supernatant stained with calcofluor; (F, G) B3501 + NET-enriched supernatant labeled with FUN1; (H) Overlay of F and G images. Bars = 20 µm. Human neutrophils were incubated in the presence or absence of PMA, capsular B3501 or acapsular CAP67 yeasts. Supernatants were added to cultures of wild-type B3501 yeasts in the presence or absence of DNAse (I), myeloperoxidase inhibitor (J), elastase inhibitor III (K), collagenase I inhibitor (L) and anti-histone neutralizing antibody (M) and growth/viability was evaluated as colony-forming units (CFU) relative to the control. The data are presented as the mean CFU ± SD of 4 donors performed in triplicate. +p<0.05 compared with unstimulated control (PBS-CTR or DMSO); *p<0.05 compared with PMA.