Fig 1.
The killing activity of neutrophils against different strains of P. insidiosum zoospores.
(A) Neutrophils from healthy donors (n = 6) were incubated with opsonized zoospores for 2 h. The percentage of colony counts on blood agar from each strain was calculated and compared with the same strain without neutrophils (100%). (B) The viability of zoospores after co-culture with or without neutrophils was observed using trypan blue staining under a light microscope, 400x magnification. The percentage of zoospore viability was calculated and compared with the same strain without neutrophils (100%). (*** p < 0.001, * p < 0.05).
Fig 2.
Phagocytosed zoospores from six strains of P. insidiosum by human neutrophils (n = 6).
(A) Representative flow cytometry result of gated neutrophils after co-culture with zoospores. (B) Representative histogram pictures of gated neutrophils and PE-positive cells indicating phagocytosed zoospores of P. insidiosum and gated neutrophils pretreated with or without cytochalasin D (negative control). (C) The percentages of phagocytosis after neutrophils incubated with zoospores compared with cytochalasin D treatment in each strain. (** p < 0.01, * p < 0.05).
Fig 3.
The NET counts and the levels of cell-free DNA when human neutrophils (n = 6) were treated with zoospores from six strains of P. insidiosum.
(A) Representative fluorescence images captured by confocal microscopy (original magnification 630X and scale bars indicated object size as labeled 20 μm) of neutrophils co-cultured with PMA (100 ng/ml, as a positive control) or zoospores from CBS 777.85 strain, stained with DAPI (blue), neutrophil elastase (green) and myeloperoxidase (red), and merged images for NET identification. (B) Representative pictures of NETs released by neutrophils per 100 cells under a fluorescence microscope (n = 6 for all groups), stained with DAPI (blue), 400x magnification. (C) The percentages of NET-released neutrophils compared with negative control (unstimulated neutrophils) counted NETs per 100 neutrophils. (D) The levels of cell-free DNA in supernatants from neutrophils co-cultured with zoospores. (*** p < 0.001, ** p < 0.01).