Journal: Veterinary Research

Article Title: Evaluation of the innate immune response of caprine neutrophils against Mycobacterium avium subspecies paratuberculosis in vitro

doi: 10.1186/s13567-023-01193-7

Figure Lengend Snippet: Gating strategy used for phagocytosis quantification. A Neutrophils, B Neutrophils with Map -GFP. In the first column, cells were gated based on FSC and SSC; in the second column, a doublet discrimination strategy was applied; in the third column, neutrophils were gated by myeloperoxidase positivity by fluorescence in the APC-Vio770 channel; in the fourth column, GFP + neutrophils were gated in the FITC channel and considered positive to phagocytosis of Map- GFP. Uninfected, non-incubated cells were used to ensure cell integrity and purity. Uninfected, incubated cells were used to adjust the neutrophil gate based on scatter parameters. This sample was also used to adjust the fluorescence thresholds of GFP and AF750. Flow cytometry data from 10 000 neutrophils from each animal ( n = 3) was analyzed using MACSQuantify™ software (Miltenyi Biotec Inc).

Article Snippet: After two washes with PBS, an overnight incubation with both the rabbit anti-MPO polyclonal Alexa Fluor 750 Conjugated (AF750) (BS-4943R-A750, Bioss, Woburn, MA, USA) [ ] at a 1:200 dilution, and the mouse anti-pan-histone primary antibody (MAB3422, Merck, Darmstadt, Germany) [ ] for NETs detection, at a 1:400 dilution was performed.

Techniques: Fluorescence, Incubation, Flow Cytometry, Software