SiMA: A simplified migration assay for analyzing neutrophil migration

Abstract

In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and three non-asthmatic patients gives a first hint to the capability of SiMA assay in the context of migration based diagnostics. Using SiMA we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB , the effect of the matrices FN versus HEM as well as the response to different medications, that is, Prednisolone induced a change of direction of migrating neutrophils in FN but no such effect was observed in human placental matrix. In addition, neutrophils of asthmatic individuals showed an increased proportion of cells migrating toward the vehicle. With the SiMA platform we presented a simplified but yet flexible platform for cost-effective tracking and quantification of neutrophil migration. The introduced method is based on a simple microscopic video stage, standardized, commercially available, µ-fluidic migration chambers and automated image analysis, and track validation software. © 2017 International Society for Advancement of Cytometry.

Tumstatin fragment selectively inhibits neutrophil infiltration in experimental asthma exacerbation

“…To assess the in vitro inhibitory effect of CP17 on the migration of neutrophils, we used the SiMA migration assay described in . In this assay, an IL‐8 and a fMLP gradient induce a chemotaxis, that is a directed movement of the neutrophiles towards the chemokines.…”

“…The assay was performed as previously described . In brief, for time‐lapse imaging of neutrophil migration, neutrophils were placed into a μ‐slide Chemotaxis 3D (IBIDI, Martinsried, Germany) and attracted towards IL‐8 or fMLP.…”

“…All image data were collected on a USB flash drive and analysed using the open source migration analysis published in implemented in MATLAB (R2016a, Mathworks, USA). This software uses a background subtraction and texture‐based image segmentation to detect neutrophil granulocytes.…”

“…To assess the quality of the tracking results, we followed the scheme presented in Ref . and calculated the valid observation time (VOT) which is defined as the length of all valid trajectories divided by the sum of the length of all valid and invalid trajectories.…”

Moving into a new dimension: Tracking migrating cells with digital holographic cytometry in 3D

“…These assays have been frequently used to study movement of cells. Weckmann et al developed an interesting system to monitor and quantify neutrophils on single cell level, by utilizing commercially available tissue culture hardware, simple video microscopic equipment, and standardized tracking (7). However, the interest for novel standardized real-time analyses using quantitative phase imaging (QPI) for cell tracking is growing (Fig.…”