Understanding Macrophage Function With Real-Time Live-Cell Analysis

Confirm differentiation. Monitoring morphological changes during differentiation of immortalized THP-1 cells and blood derived primary monocytes.  THP-1 cells were differentiated into M0 macrophages by exposing the cells to 5 ng/mL PMA for 48 hours. Primary monocytes were differentiated into either M1-like phenotype, by incubating in 50 ng/mL GM-CSF for 6 days followed by 1 ng/mL LPS + 20 ng/mL IFN-γ for one more day, or into M2- like phenotype, by incubating in 50 ng/mL M-CSF for 6 days followed by 20 ng/mL IL-4 for one more day. Note the morphology is consistent with previous findings (Young et al, J Immunol, 1990); the M1 population have fried egg morphology, while M2 have a mixed population of fried egg- and spindle-shaped cells.

Track differentiation. Phase images show morphology of differentiating human iPSC-derived monocytes (Day 0-7) which produce terminally differentiated human iPSC-Derived Macrophages containing large vesicles. Differentiation was achieved by adding 100 ng/mL M-CSF to maintenance media for at least 7 days.
 

Visualize morphology and cell surface markers, to study differentiation. THP-1 monocytes were exposed to media (undifferentiated), vitamin D3 or PMA in the presence of Incucyte^® Fabfluor-488 antibody complexed to CD11b, CD14 or CD40. PMA showed a marked change in cell morphology (HD-phase contrast images) compared to media alone or vitamin D3 treated cells. The kinetic graphs highlight differential and time-dependent surface protein expression in response to the various treatments.

Biological significance of monocyte maturation. THP-1 cells, either naïve or PMA differentiated M0 were seeded on ClearView chemotaxis membranes coated with fibronectin. Cells were then exposed to a C5a gradient and images acquired every hour using the Incucyte system. Analysis of pharmacological response was performed at t=30 hr. While differentiated macrophage-like THP-1 cells had a strong concentration dependent response towards C5a, little or no response was observed in THP-1 cells prior to differentiation.

Importance of kinetic monitoring of macrophage chemotaxis. Primary blood monocytes were differentiated into M2-like phenotype; 50 ng/mL M-CSF for 6 days followed by 20 ng/mL IL-4 for a further day. Macrophages were seeded on ClearView chemotaxis membranes coated with Matrigel, then exposed to a C5a gradient and images acquired every 2 hours using the Incucyte system. Bottom-side phase analysis was performed and pharmacological response determined at 4.5 and 11 hrs. Note a substantial time-dependent C5a potency shift due to delay in response at higher concentrations, a specific response as this was not observed when neutrophils were used.

Quantification of phagocytosis. iPSC-derived macrophages (Axol Bioscience) engulf Incucyte^® pHrodo^® Green E. coli Bioparticles^® in a time- and cell-number-dependent manner.  Phase contrast and fluorescence blended images of iPSC-derived macrophages in the presence 100 ug/well of pHrodo^® Green E. coli Bioparticles^® illustrate the uptake of Bioparticles^® over time.  Below each is the corresponding masked-image highlighting the use of segmentation to fully quantify the phagocytosis kinetics.  The time-course of iPSC-derived macrophage phagocytosis using a single pHrodo^® Green E. coli Bioparticle amount (100 ug/well) shows cell –number-dependence.

Anti-CD47 induces phagocytosis of CCRF cells. Labeled CCRF-CEM cells treated with increasing concentrations of anti-CD47 antibody (B6H12.2) and added to human bone marrow-derived macrophages (BMDM; values shown are mean ± SEM, n=4).

Imaging and quantification of engulfed cells inside macrophages. Engulfed Jurkat cells inside J774.A1 macrophages display high fluorescence intensity, proportional to the amount of cell phagocytosis. Rapid imaging and 2 min intervals can be used to create movies showing individual engulfment events.

Differentiation determines phagocytic capability. THP-1 cells and were differentiated into M0, M1 or M2 macrophages by exposing the cells to 200 nM PMA for 24 hr, 200 nM PMA for 24 hr + 20 ng/mL IFNγ + 1 µg/mL LPS, or 200 nM PMA for 24 hr + 20 ng/mL IL-4, respectively. Cells were then co-cultured with pHrodo red labelled apoptotic Jurkats and the phagocytic capability of differentiated macrophages was assessed by fluorescent signal of engulfed Jurkats. Data shows that both M0 and M2 differentiated THP-1 cells have significantly higher phagocytic capability, which is consistent with the anti-inflammatory function of M2 macrophages.