RESUMO
Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.
Assuntos
Ensaios de Migração de Leucócitos/métodos , Quimiotaxia de Leucócito/imunologia , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Neutrófilos/imunologia , Doença Pulmonar Obstrutiva Crônica/sangue , Ensaios de Migração de Leucócitos/instrumentação , Fatores Quimiotáticos/química , Humanos , Microfluídica/instrumentação , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/química , Doença Pulmonar Obstrutiva Crônica/imunologia , Escarro/químicaRESUMO
Cell migration on two-dimensional (2D) substrates follows entirely different rules than cell migration in three-dimensional (3D) environments. This is especially relevant for leukocytes that are able to migrate in the absence of adhesion receptors within the confined geometry of artificial 3D extracellular matrix scaffolds and within the interstitial space in vivo. Here, we describe in detail a simple and economical protocol to visualize dendritic cell migration in 3D collagen scaffolds along chemotactic gradients. This method can be adapted to other cell types and may serve as a physiologically relevant paradigm for the directed locomotion of most amoeboid cells.