Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays

Abstract

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays. (Blood. 2012;120(14): e45-e53)

Characterizing asthma from a drop of blood using neutrophil chemotaxis

“…For example, we have previously used a similar chemotaxis technology to analyze neutrophils obtained from an arthritic mouse model (53), a disease where neutrophil inflammation is known to regulate dysfunction (54). In this study we observed a ∼10-fold increase in neutrophils from arthritic mice adhering to the substrate, a significant decrease in neutrophil directionality, and no significant difference in chemotaxis velocity compared with wild-type mice (31). Importantly, the neutrophil defect observed in this prior study differs significantly from the asthmatic neutrophil phenotype characterized in this work, suggesting that differences in neutrophil function could be observed to differentiate other inflammatory disorders from asthma.…”

“…We adapted a microfluidic neutrophil chemotaxis platform that we previously developed (31) to assay the neutrophil function of mildly asthmatic and nonasthmatic patients in a clinical setting. Briefly, the diagnostic chip has two primary components: (i) a functionalized migration channel in the base, where the rapid neutrophil purification and chemotaxis are performed, and (ii) a multifunction lid that houses all of the reagents required for the diagnostic test and mitigates evaporation, which can be detrimental for long-term open microfluidic experiments (32).…”

“…1B, Fig. S1, and Dataset S1) (31). Following a time-lapse capture of the neutrophil chemotaxis, the data are automatically tracked and analyzed using custom tracking software, dubbed Je'Xperiment (JEX), eliminating the need for onerous manual cell tracking ( Fig.…”

Mkit: A cell migration assay based on microfluidic device and smartphone

“…Many of these devices can configure chemical gradients without the requirement of external fluid delivery instruments (Ge et al 2015;Sackmann et al 2012;Wu et al 2016). Recently, new microfluidic methods have been developed to allow rapid immune cell chemotaxis test directly from a drop of whole blood by incorporating on-chip cell isolation module (Agrawal et al 2008;Hamza and Irimia 2015;Jones et al 2016;Sackmann et al 2012;Sackmann et al 2014b;Wu et al 2016). Several studies have also employed microfluidic cell migration systems for disease orientated applications (Butler et al 2010;Jones et al 2014;Wu et al 2015).…”

“…Over the past near two decades, microfluidic devices have become widely used research tools for quantitative cell migration and chemotaxis studies owing to their ability to better control microenvironments, low reagents consumption, simple and automated fluid handling systems (Huang et al 2009;Jeong et al 2010;Jeong et al 2013;Li et al 2016;Sackmann et al 2014a;Vargas et al 2014;Wu et al 2013). Many of these devices can configure chemical gradients without the requirement of external fluid delivery instruments (Ge et al 2015;Sackmann et al 2012;Wu et al 2016). Recently, new microfluidic methods have been developed to allow rapid immune cell chemotaxis test directly from a drop of whole blood by incorporating on-chip cell isolation module (Agrawal et al 2008;Hamza and Irimia 2015;Jones et al 2016;Sackmann et al 2012;Sackmann et al 2014b;Wu et al 2016).…”