Figure 2.

BacLight fluorescence to investigate intracellular MAP viability. (A) Shows one representative image of monocyte derived macrophage culture without vitamin D₃ treatment from a clinical cow. (B) SYTO 9 (yellow) represents viable intracellular MAP detected in the 488 nm channel. (C) Propidium iodide (P.I.) was used to label intracellular nonviable MAP in the 561 nm channel (magenta). Large, dense magenta spheres are nuclei and were excluded from analysis based on size. (D) Extracellular MAP (cyan) that was not washed away was detected in the 640 nm channel, and its dual labeling with P.I. or SYTO 9 flagged those bacteria for exclusion from analysis.