An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

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.

Chemotaxis, a process of directed cell migration to soluble chemical concentration gradient, is critically involved in many biological processes including immune response^1,2,3, tissue development⁴ and cancer metastasis⁵. Neutrophils are the most abundant white blood cell subset and play crucial roles in enabling the body's innate host defense functions, as well as in mediating adaptive immune responses^6,7. Neutrophils are equipped with highly-regulated chemotactic ma....

All human sample collection protocols were approved by the Joint-Faculty Research Ethics Board at the University of Manitoba, Winnipeg.

1. Microfluidic Device Fabrication (Figure 1A)

1. Design and print transparency mask.
   1. Design the device as detailed previously²⁵. See Figure 1A.
      NOTE: The device includes two layers. The first layer (4 µm high) defines the cell.......

Neutrophils are negatively selected from a drop of whole blood directly in the microfluidic device. The purity of the isolated neutrophils was verified by on-chip Giemsa staining and the results showed the typical ring-shaped and lobe-shaped nuclei of neutrophils (Figure 2A)²⁵. This indicates an effective on-chip neutrophil isolation at high purity from a small volume of whole blood. Furthermore, the docking structure can effectively a.......

In this paper, a detailed protocol to directly isolate neutrophils from whole blood followed by the chemotaxis test, all on a single microfluidic chip, was described. This method offers useful features in its easy operation, negative selection of high purity neutrophils, rapid sample-to-result chemotaxis test, reduced reagents and sample consumption, and accurate cell migration data analysis. As a rough estimate, at least 25% of the neutrophils from the input whole blood sample effectively entered the docking structure i.......

This work is in part supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). We thank the Clinical Institute of Applied Research and Education at the Victoria General Hospital in Winnipeg and Seven Oaks General Hospital in Winnipeg for managing clinical samples from human subjects. We thank Dr. Hagit Peretz-Soroka for helpful discussion about the assay operation strategies. We thank Professor Carolyn Ren and Dr. Xiaoming (Cody) Chen from the University of Waterloo for their....