Macrophages can exhibit both protective and pathogenic phenotypes in response to environmental cues. Evaluating these distinct functional phenotypes is essential for understanding host defense, auto-immunity, antitumor immunity as well as anti-inflammatory function.
The IncuCyte® live-cell analysis system enables functional analysis of macrophages through applications such as phagocytosis and chemotaxis. Images and movies enable detailed assessment of macrophage morphology.
Verify differentiation. Immortalized THP-1 cells and blood derived primary monocytes were differentiated on fibronectin or Matrigel coated TC-treated flasks, respectively. 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 that the M1 population have fried egg morphology, while M2 have a mixed population of fried egg – shaped and spindle – shaped cells. This difference in morphology in M1 vs. M2 cells is consistent with previous observations (Young et al., The Journal of Immunology, 1990).
Biological significance of monocyte maturation. Immortalized THP-1 cells or PMA differentiated M0 (macrophage-like THP-1 cells) were seeded on ClearView chemotaxis membranes coated with fibronectin. Cells were then exposed to a C5a gradient at the indicated concentrations. Images were acquired every 60 minutes using the IncuCyte system and phase analysis was performed. 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, no response was observed in THP-1 cells prior to differentiation, suggesting biological significance of monocyte maturation in C5a-mediated chemotaxis.
Importance of kinetic monitoring of macrophage chemotaxis. Primary blood monocytes were differentiated into M2- like phenotype, by treatment with 50 ng/mL M-CSF for 6 days followed by 20 ng/mL IL-4 for one more day. Macrophages were seeded on ClearView chemotaxis membranes coated with Matrigel, then exposed to a C5a gradient at the indicated concentrations (A). Images were acquired every 2 hours using the IncuCyte system and bottom-side phase analysis was performed. Analysis of pharmacological response was performed at t=4.5 (B) and 11 hrs (C), noting a significant EC50 value changed depending upon time of analysis due to delay in macrophage response to 1000 and 333 nM C5a. The observed delay with higher concentration of C5a was specific to Macrophage cells. When same concentrations were used in with Neutrophil cells, the delay was not detected (data not shown).
Differentiation determines phagocytic capability. Immortalized 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 (A).