Fig. 3. Erythrophagocytosis is substantially elevated in TB mice.

(A to D) Validation of the erythrophagocytosis assay using ImageStream®. Gates for PKH26^low, PKH26^med, and PKH26^high were set according to the fluorescence of the PKH26-labeled RBCs (A), and phagocytes were analyzed accordingly (B). The rate of internalization of PKH26^med (yellow) and PKH26^high (red) cells was determined (a score higher than 0 is considered internalized), and the three different populations were visualized (D). Only PKH26^high phagocytes show complete internalization and engulfment of entire RBCs. These plots and images are from the erythrophagocytosis experiment in which RBCs from TB mice were coincubated with healthy Ctrl splenocytes. (E) Representative flow cytometry plots with the gating strategy for F4/80^highCD11b^low red pulp macrophages, F4/80^highCD11b^high macrophages, and F4/80^lowCD11b^high monocytes/neutrophils (gated on CD45⁺ splenocytes). (F and G) PKH26 fluorescently labeled healthy RBCs were coincubated with splenocytes (spl) of either Ctrl or TB mice. Representative histograms of PKH26⁺ phagocytes including PKH26^med and PKH26^high (F) and the quantification of the percentage PKH26^high cells representing phagocytes that engulfed complete RBCs (G). (H and I) PKH26 fluorescently labeled RBCs from Ctrl or TB mice were coincubated with healthy splenocytes. Representative histogram of PKH26⁺ F4/80^lowCD11b^high monocytes/neutrophils (H) and the quantification of the percentage PKH26^high cells representing phagocytes that engulfed complete RBCs (I). Groups (n = 5 to 6 per group) were compared by two-tailed unpaired t tests with Welch’s correction and data are represented as means + SEM. Asterisks indicate differences between Ctrl and TB group. *P < 0.05 and ***P < 0.001.