A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Abstract

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Actin dynamics shape microglia effector functions

“…(3) In general agreement with earlier studies in macrophages, impaired actin dynamics lead to reduced microglia chemotaxis, proliferation and phagocytosis (e.g. de Oliveira and Mantovani 1988; Allen et al 1997Allen et al , 1998Jonsson et al 2012;Iqbal et al 2013). The actin cytoskeleton is sensitive to oxidative stress, which may lead to oxidation of exposed cysteine residues, disulphide bonds and, ultimately, reduced dynamic cytoskeletal plasticity (Bencsath et al 1996;Dalle-Donne et al 2001;Haarer and Amberg 2004).…”

A Real-Time Assay for Neutrophil Chemotaxis

“…These variations most likely reflect individual differences in basal activation of neutrophils and biological specificities between donors. Similar observations have been reported with human monocytes when using the xCELLigence instrument (28). These results emphasize the importance of real-time assays, without which early or delayed signals could be missed.…”

“…Optimal results in terms of maximal slope values and sensitivity to increasing concentrations of IL-8 were obtained with 4 10 6 cells/mL (Figure 2B). This cell density is similar to that used in other migration assays with human neutrophils (6 10 6 cells/mL) (21) and equine neutrophils (3 10 6 cells/mL, 2 10 6 cells/mL) (31, 37), and in other impedance-based assays with monocytes and macrophages (8 10 6 cells/mL) (28). Interestingly, increasing concentrations of fibronectin were not associated with higher slope values (i.e., increased neutrophil chemotaxis) (Figure 2C).…”

“…Slope analysis was performed on CI curves at points where the maximum value was reached. Iqbal and colleagues previously validated this analysis method when they examined chemotaxis of different leukocyte populations with the xCELLigence instrument and a scanning electron microscope at different time points (28). Optimal results in terms of maximal slope values and sensitivity to increasing concentrations of IL-8 were obtained with 4 10 6 cells/mL (Figure 2B).…”

“…Compared to other ECIS systems, xCELLigence, developed by ACEA Biosciences in partnership with Roche Applied Science, allows easier and faster experimental analysis with user-friendly software without the need for lengthy preparation and stabilization steps. This system has been validated for analysis of macrophage and monocyte chemotaxis (28), but has yet to be adapted for neutrophils.…”