A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

Abstract

Source: Yang, K., et al. An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood. J. Vis. Exp. (124), (2017)

This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic chip.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Microfluidic device preparation. Microfluidic Cell Migration Assay Preparation Prepare 50 µg/mL fibronectin solution by diluting 50 µL of stock fibronectin solution (1 mg/mL) to 950 µL Dulbecco's phosphate-buffered saline (DPBS) inside a biosafety cabinet. Prepare the mig…

Representative Results

Figure 1: Illustration of the all-on-chip method for neutrophil chemotaxis analysis. (A) Illustration of the microfluidic device. The device includes two layers. The first layer (4 µm high) defines the cell docking barrier channel to trap the cells beside the gradient channel. The second layer (60 µm high) defines the gradient generating channel, the port and channel fo…

Materials

Device fabrication
Mask aligner	ABM	N/A
Spinner	Solitec	5000
Hotplate	VWR	11301-022
Plasma cleaner	Harrick Plasma	PDC-001
Vacuum dessicator	Fisher Scientific	08-594-15A
SU-8 2000 thinner	Microchem	SU-8 2000
SU-8 2025 photoresist	Microchem	SU-8 2025
(tridecafluoro-1,1,2,2- tetrahydrooctyl) trichlorosilane	Gelest	78560-45-9
Si wafer	Silicon, Inc	LG2065
Glass slides	Fisher Scientific	12-544-4
Cutting pad	N/A	N/A	Custom-made
Punchers
On-chip cell isolation and chemotaxis assay
RPMI 1640	Fisher Scientific	SH3025502
DPBS	Fisher Scientific	SH3002802
Bovine serum albumin (BSA)	Sigma-Aldrich	SH3057402
Fibronectin	VWR	CACB356008
fMLP	Sigma-Aldrich	F3506-10MG
Magnetic disks	Indigo Instruments	44202-1	5 mm in diameter, 1 mm thick
FITC-Dextran	Sigma-Aldrich	FD10S
Rhodamine	Sigma-Aldrich	R4127-5G
Giemsa stain solution	Rowley Biochemical Inc	G-472-1-8OZ
EasySep Direct Human Neutrophil Isolation Kit	STEMCELL Technologies Inc	19666
Dithiothreitol	Sigma-Aldrich	D0632
Nikon Ti-U inverted fluorescent microscope	Nikon	Ti-U
Microscope environmental chamber.	InVivo Scientific	N/A
CCD camera	Nikon	DS-Fi1