Identification of labeled and infected labeled monocytes-macrophages using flow-cytometry from A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors

The antibody allowing identification of monocytes-macrophages required a secondary Alexa FluorTM 488 conjugated anti-mouse antibody. Flow cytometric analyses were performed with a BD LSR FortessaTM X-20 (BD Biosciences, San Jose, CA, USA). BD FACSDivaTM software (v 8.0.2) was used to analyze the cytometric data. For each sample, dot plots were analyzed. Debris was removed on the basis of morphological criteria, regions were defined on the basis of uninfected control samples and isotype-control staining. The intensity of green fluorescence (Alexa FluorTM 488) was on the vertical axis, plotted against the intensity of red fluorescence (TurboFP650) on the horizontal axis. Labeled cells emitting a green fluorescence were detected in the upper part of the graph. Infected labeled cells emitting both types of fluorescence (green and red) were revealed by dots in the upper right-hand part of the graph. Unlabeled infected cells could also be seen in the lower right-hand part of the graph. Results were expressed as a percentage. Some examples are presented. Staining in the gall bladder is shown for the monocytes-macrophages.