PMC

Long term E. coli phagocytosis is biphasic, whereas S. aureus uptake reaches a plateau at 4 h. (A) and (B) BMDMs from male C57BL/6J mice were seeded at 50,000, 12,500 and 3125 cells per well and incubated with 200 μg/ml green E. coli (A) or red S. aureus (B) bioparticles. Fluorescence signal was measured in the IncuCyte imaging platform every 20 min for 24 h at 37 °C. Data are presented as mean ± SEM from 3 biological replicates in technical duplicates.

BMDM phagocytosis is pathogen particle number and cell density-dependent. (A) BMDMs (10⁵) from male C57BL/6J mice were incubated with green E. coli bioparticles (1 mg/ml to 1 μg/ml) and fluorescence emission was measured in the IncuCyte imaging platform every 10 min for one hour at 37 °C. (B)–(D) Representative images from one experiment at one hour. (E) BMDMs (100,000–1562 cells per well) were incubated with 200 μg/ml green E. coli bioparticles for one hour at 37 °C and fluorescence emission was measured in the IncuCyte imaging platform with 10 min intervals. (F) RAW264.7, BV-2, THP-1 and hiPS cell-derived macrophages (5 × 10⁴) were incubated with 200 μg/ml green E. coli bioparticles and fluorescence emission was measured in the IncuCyte imaging platform every 10 min for one hour at 37 °C. (G) Side-by-side comparison of green E. coli bioparticle phagocytosis by 100,000 BMDMs measured with a spectrofluorometer and the IncuCyte system. Data are presented as mean ± SEM from 3 to 4 biological replicates in technical duplicates, apart from F and G where data are from one experiment with two technical replicates and are presented as mean ± SD. Scale bar 100 μm.

Macrophage polarization status affects phagocytic capacity. (A) BMDMs (2 × 10⁵) from C57BL/6J male mice were polarized by incubation with vehicle, 100 ng/ml LPS, 20 ng/ml IFN-γ, 20 ng/ml IL-4 or 10 ng/ml IL-10 at 37 °C for 16 h and (A) Nos2, (B) Arg1 and (C) Mrc1 mRNA expression was measured. (D) Polarized BMDMs (5 × 10⁴) from C57BL/6J male mice were subsequently incubated with 200 μg/ml green E. coli bioparticles for one hour. Fluorescence emission was measured in the IncuCyte imaging platform for one hour every 10 min. (E) Time kinetics of the macrophage polarization experiments. Data are presented as mean ± SEM from 4 biological replicates in technical duplicates. Statistical analysis was carried out with one-way ANOVA with Dunnett’s multiple comparisons post hoc test.

Pre-treatment with LPS leads to reduced E. coli phagocytosis by macrophages. (A) and (B) BMDMs (5 × 10⁴) were primed with a range of LPS doses for 16 h and cells were incubated for another 24 h at 37 °C in culture media, after which 200 μg/ml green E. coli bioparticles were incubated with the macrophages in the IncuCyte platform for another hour. Green object counts in vehicle-treated macrophages were set to 100% and object counts in LPS pre-treated macrophage groups within the same experiment were normalized to demonstrate% reduction compared to the control. (C) Representative images from one experiment at one hour. Data from 5 biological replicates in technical duplicates are presented as mean ± SEM. Statistical analysis was carried out with Kruskal-Wallis and Dunn’s multiple comparisons post hoc test. Scale bar is 100 μm.

Macrophage phagocytosis is inhibited by actin and microtubule inhibitors and enhanced by opsonization. (A)–(D) BMDMs (5 × 10⁴) from C57BL/6J male mice were pre-treated with the actin and microtubule inhibitors Cytochalasin D and Nocodazole at the indicated concentration for one hour and were then incubated with 200 μg/ml green E. coli bioparticles at 37 °C for another hour. (E) and (F) BMDMs (5 × 10⁴) from C57BL/6J male mice were pre-treated with the vacuolar H⁺-ATPase inhibitor Bafilomycin A1 at the indicated concentration for one hour and were incubated with 200 μg/ml green E. coli bioparticles at 37 °C for another hour. (G) and (H) 200 μg/ml green E. coli bioparticles were opsonized with murine IgG or diluted human serum for 30 min at 37 °C and were then administered to 50,000 BMDMs from C57BL/6J male mice for one hour. (A), (C), (E) and (G) demonstrate the kinetics of phagocytosis at 10 min intervals. Fluorescence emission from all experiments was measured in the IncuCyte imaging platform and statistical analysis was carried out with one-way ANOVA with Dunnett’s multiple comparisons post hoc test. Data are presented as mean ± SEM from 3 to 5 biological replicates in technical duplicates.

Fluorescent object segmentation analysis by the IncuCyte Basic Software. BMDMs (10⁵) from male C57BL/6J mice were incubated with 1 mg/ml (A) or 10 μg/ml (B) green E. coli bioparticles and fluorescence emission was measured in the IncuCyte imaging platform for one hour at 37 °C. Representative images showing the green channel signal (left), the green object mask which was applied for quantification (middle) and the overlay of the two (right). (C) The edge split setting in the IncuCyte Basic Software allows for quantification of closely-spaced objects in both fluorescence channels. Images from 100,000 BMDMs incubated with 1 mg/ml green E. coli bioparticles for one hour at 37 °C without (left) or with (right) edge split. The magenta lines delineate separate objects. Scale bar 100 μm. BMDMs (2.5 × 10⁴ per well) were incubated with an equal mix of 200 μg/ml red pHrodo S. aureus bioparticles and 200 μg/ml FITC-conjugated Zymosan A (D) or FITC-conjugated 2 μm latex beads (E) and phagocytosis was studied at one hour using confocal microscopy. White arrows indicate FITC-conjugated Zymosan A or latex beads on the macrophage membrane, orange arrows indicate unphagocytosed FITC-conjugated Zymosan A or latex beads and yellow arrows indicate unphagocytosed S. aureus bioparticles. NucBlue Live ReadyProbes reagent was used for nuclei staining in (D). Images are from one representative of three independent experiments. Scale bar 20 μm.

Primary murine macrophage populations exhibit differential efficiency of phagocytosis of different pathogens. C57BL/6J murine lungs and peritoneal cavities were lavaged and cells were lysed to remove red blood cells. (A) Alveolar macrophages were defined as Siglec F⁺ and CD11c⁺ cells within the CD45⁺ cell population of total lung cells. (B) Peritoneal resident macrophages were defined as F4/80⁺ and CD11b⁺ cells within the CD45⁺ cell population of total peritoneal cavity cells. (C) and (D) Macrophage numbers were inferred from total numbers and the Flow Cytometry gating strategy. (E) Alveolar, peritoneal and BMDMs (12,500 cells) were treated with 200 μg/ml green sterile Zymosan, E. coli and red S. aureus bioparticles for four hours and fluorescence emission was measured in the IncuCyte imaging platform every 15 min. (F)–(H) Kinetics of pathogen phagocytosis; 12,500 alveolar (F), peritoneal (G) and murine BMDMs (H) over four hours measured in the IncuCyte platform every 15 min. Data at four hours are presented as mean ± SEM from 4 biological replicates in technical duplicates. Statistical analysis was carried out with a two-way ANOVA and Tukey’s post hoc multiple comparisons test.