Measuring neutrophil function with real-time live-cell analysis
Neutrophils are the first line of defense at the site of an infection, playing an essential role in the innate immune system, by both ingesting bacteria and releasing antimicrobial enzymes that degrade and kill pathogens. Neutrophil recruitment and function are critical for host defense, but can contribute to inflammation , causing progression of diseases such as Alzheimer’s and rheumatoid arthritis.
The IncuCyte® live-cell analysis system has been used to evaluate neutrophil response to chemokines using the chemotaxis assay, as well as assessing cell function via in vitro NETosis, phagocytosis and differentiation assays.
Measure relevant surface contact-mediated cell migration. The low pore density of the ClearView membrane ensures that cells must migrate across the biologically relevant surface towards the chemoattractant. Neutrophils seeded on an uncoated ClearView membrane were unable to migrate towards the chemoattractants IL-8 and fMLP (left); however, those on Matrigel-coated membranes showed clear chemotactic profiles (right). These data suggest that integrin and/or cell surface receptor interactions with the substrate play a key role in neutrophil chemotaxis in this model. In contrast, no coatings were required for neutrophil migration studies using the Corning® Transwell® consumable (data not shown), suggesting that active migration of neutrophils across the Transwell® filter is absent in the Corning® Transwell® system.
Visualising & quantifying NETosis
Visualising and quantifying NETosis. HL-60 cells were differentiated to neutrophil-type using DMSO (1.25% v/v) and ATRA (0.1 μM). The resulting multinuclear dHL-60 cells were stimulated using PMA. At ~2 hours post stimulation, nuclei begin to decondense. HD phase and fluorescent images show characteristic morphology of neutrophil NETosis, as the cytoplasm mixes with karyoplasm the nuclei are no longer visible in Phase images. The nuclear contents are moved to the plasma membrane and released.. Kinetic data shows PMA induced NETosis as detected by IncuCyte® Cytotox Green reagent binding to external DNA.
Differentiation determines phagocytic capability. HL-60 cells were differentiated into neutrophils by exposing the cells to 0.1 µM atRA and 1.2% DMSO for 5 days. Cells were then seeded and IncuCyte® pHrodo® Green Bioparticles were added. The phagocytic capability of differentiated HL-60 cells was assessed. Data shows a striking differential between naïve and differentiated HL-60 cells, with the differentiated cells yielding a substantial increase in fluorescence area, indicative of phagocytosis.
Phagocytosis by primary neutrophils. Primary neutrophils extracted from peripheral blood were then seeded and IncuCyte® pHrodo® Green Bioparticles were added. Primary neutrophils are highly phagocytic, engulfing bioparticlesleading to an increase in fluorescent signal.
Track differentiation. Phase images show differences in morphology of undifferentiated HL-60 cells and HL-60 cells differentiated to neutrophil-like cells by adding 1.25 % DMSO and 1 µM ATRA to RPMI + 10% FBS media. Kinetic data shows differences in cell proliferation as detected by HD phase confluence.
Monitoring Changes in Cell Shape
Changes in cell shape (eccentricity) of PMNs. PMNs from whole blood seeded in Matrigel with increasing concentration of CSCL8 with IncuCyte FabFluor-488 labeled CD11b antibody and Opti-green background suppressor, with imaging in the IncuCyte Live-Cell Analysis System and phase and green fluorescence images captured. IncuCyte Cell-by-Cell Analysis Software used to quantify changes in cell shape and CD11b expression over time.
1. NETosis; Gupta et al 2017. J Immunol. 2017 Dec 1. pii: ji1700905. doi: 10.4049/jimmunol.1700905. [Epub ahead of print]