Functional analyses of the neutrophil subpopulations.

A) Neutrophils were either fixed immediately after separation (Fresh) or allowed to enter apoptosis spontaneously (-FasL) or through the Fas pathway (+FasL) for 4 or 20 h, after which they were fixed and stained using a TUNEL assay in combination with immunostaining for OLFM4. The histogram shows fresh (blue) and apoptotic (20 h +FasL, red) neutrophils stained using TUNEL. The dot plot shows double staining of fragmented DNA (TUNEL) and OLFM4, with the quadrants set using fresh neutrophils for TUNEL and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of apoptosis +SD, based on TUNEL-positivity, in the OLFM4-positive (black) and -negative (grey) subpopulations of three donors. B) Freshly isolated and apoptotic neutrophils (20 h incubation without or with FasL) were subjected to immunostaining of OLFM4 (black) and NGAL (grey). The diagram shows the mean percentage of positive cells +SD for each protein from three independent experiments. C) Neutrophils were allowed to phagocytose M. tuberculosis H37Ra strain expressing GFP (M.tb-GFP), either unopsonized or serum-opsonized, at an MOI of 5 for 30 min, fixed, and immunostained for OLFM4. The histogram shows neutrophils incubated without (No prey) or with (+M.tb-GFP ops) serum-opsinized M. tuberculosis. The dot plot shows fluorescence intensity in the GFP and OLFM4 channels after phagocytosis of opsonized bacteria, with the quadrants set using neutrophils with no prey added for M.tb-GFP and control (omitted primary antibody) for OLFM4. The bar graph shows the mean percentage of phagocytosing neutrophils +SD in the OLFM4-positive (black) and -negative (grey) subpopulations from three independent experiments.