Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay.

Abstract

Neutrophils are the most common leukocyte in human blood and are the first cells to respond to injury and infection. Improper neutrophil chemotaxis can have deleterious effects on human health, including autoimmune diseases, poor innate immune response, and cancer. Therefore, gaining a better understanding of the signaling pathways governing chemotactic responses in these cells is important. One of the main challenges of working with primary human neutrophils is their short lifespan (about 1 day), making genetic manipulations not feasible. PLB-985 cells, which are pluripotent hematopoietic cells that can easily be differentiated to neutrophil-like cells, are amenable to genetic manipulations, including the expression of fluorescently tagged proteins-of-interest (POI) and gene editing using the CRISPR/CAS9 system to delete genes-of-interest (GOI). The use of PLB-985 cells can therefore greatly facilitate our understanding of the molecular mechanisms governing neutrophil biology during chemotaxis and serve as a good system to complement results gained from pharmacological inhibition of primary neutrophils. To better study the role and localization of proteins during chemotaxis, the underagarose assay has become a widely used and quantitative assay for measuring several aspects of chemotaxis. The objective of this chapter is to provide protocols for (1) the generation of genetically altered PLB-985 cell lines, (2) the set-up of an underagarose chemotaxis assay, and (3) the analysis of cell movement in chemotactic gradients from an underagarose experiment.