Neutrophil Chemotaxis in One Droplet of Blood Using Microfluidic Assays

Abstract

Neutrophils are the most abundant leukocytes in blood serving as the first line of host defense in tissue damage and infections. Upon activation by chemokines released from pathogens or injured tissues, neutrophils migrate through tissues toward sites of infections along the chemokine gradients, in a process named chemotaxis. Studying neutrophil chemotaxis using conventional tools, such as a transwell assay, often requires isolation of neutrophils from whole blood. This process requires milliliters of blood, trained personnel, and can easily alter the ability of chemotaxis. Microfluidics is an enabling technology for studying chemotaxis of neutrophils in vitro with high temporal and spatial resolution. In this chapter, we describe a procedure for probing human neutrophil chemotaxis directly in one droplet of whole blood, without neutrophil isolation, using microfluidic devices. The same devices can be applied to the study the chemotaxis of neutrophils from small animals, e.g., mice and rats.

Keywords: Blood; Chemotaxis; Microfluidics; Neutrophils; Persistence; Speed.

Fig. 1

The work flow of developing microfluidic devices for chemotaxis studies is an iterative process that may include several cycles of design, fabrication, and testing. A microfluidic design is first captured as an AutoCAD drawing. The design is implemented in SU-8 photoresist on silicon wafers using photolithography masks printed from the AutoCAD drawings. Devices are then fabricated in PDMS that is cast on the SU-8-silicon mold. The devices are then tested with whole blood samples. Depending on the initial results, the design may need to be optimized further

Fig. 2

The microfluidic device or neutrophil chemotaxis in a droplet of whole blood. (a) A macrophotography of the microfluidic device fabricated in a single well plate. (b) The device immersed in IMDM media after priming. (c) Loading of 1 µL whole blood in the device using a fine pipette tip. The scale bars are 5 mm

Fig. 3

Geometrical details of the microfluidic device for neutrophil chemotaxis. (a) Bright-field microscopic image showing the microfluidic unit for characterizing neutrophil trafficking from a drop of whole blood. (b) Fluorescence microscopic image showing the chemogradient established in the unit. The scale bars are 40 µm

Fig. 4

Human neutrophil chemotaxis from whole blood. A montage of time-lapse images shows one neutrophil (nucleus stained in blue), which migrates from the whole blood in the central chamber (bottom) toward the LTB4-filled chemoattractant chamber (top). The scale bar is 40 µm. Time interval between successive frames is 2 min