This article describes a method for isolating neutrophils from whole blood samples using immunomagnetic techniques and a microfluidic device. The process allows for the study of neutrophil chemotaxis in response to chemoattractants.
To a whole blood sample, add specific antibody complexes directed against blood cells except neutrophils.
Add magnetic particles that bind to the antibody complex-bound blood cells.
Transfer the immunomagnetically tagged sample to the cell-loading port of a microfluidic chip.
Attach magnetic disks to the cell-loading port.
The magnetic field attracts the immunomagnetically-tagged cells, trapping them to the port's side walls.
The neutrophils, being untagged, flow into the chip and become trapped at the cell-docking area.
Add fluorescently-labeled chemoattractant solution and migration medium to designated reservoirs. The liquids flow through the microfluidic channels, forming a concentration gradient.
When the chemoattractant binds to the neutrophil receptor, it triggers intracellular changes, causing neutrophils to reorganize their cytoskeleton and extend pseudopods.
These pseudopods act as feet, propelling the neutrophils toward higher chemoattractant concentrations — a process called chemotaxis.
Use appropriate microscopy techniques to monitor neutrophil migration in response to the chemoattractant gradient.
Remove the deionized water from the device, and add 100 microliters of Fibronectin solution from the outlet. Wait three minutes to ensure that all the channels are filled with Fibronectin solution. Then, incubate the device in a covered Petri dish for an hour at room temperature.
Next, remove the Fibronectin solution from the device, and add 100 microliters of migration medium from the outlet. Again, wait three minutes to ensure that all of the channels are filled with the migration medium. Incubate the device for another hour at room temperature, before using the device in the chemotaxis experiment. Place 10 microliters of whole blood into a 1.5-milliliter tube. Then, add 2 microliters of the antibody cocktail in two microliters of magnetic particles from a neutrophil isolation kit, and gently mix the tube in order to magnetically label the antibody-tagged cells. Incubate the label mixture for five minutes at room temperature.
Next, attach two small magnetic disks to the two sides of the cell loading port of the device. Aspirate the medium from all ports of the device. Then, slowly pipette two microliters of the labeled blood mixture into the microfluidic device from the cell-loading port. Place the microfluidic device on the temperature-controlled microscope stage at 37 degrees Celsius. Wait a few minutes until enough neutrophils are trapped at the cell-docking area.
Next, add 100 microliters of the chemoattractant solution in 100 microliters migration medium to their designated inlet reservoirs, using two pipettors. This will generate a chemoattractant gradient by continuous laminar flow-based chemical mixing. The chemoattractant can be recombinant proteins, such as FMLP, or clinical samples, such as the supernatant of sputum from patients with COPD.
Once flow has stabilized, acquire fluorescence images of the FITC dextran gradient in the channel. Then, incubate the device on the temperature-controlled microscope stage, or in a conventional cell culture incubator for 15 minutes. Following incubation, image the gradient channel, using a 10 times objective to record the cell's final positions for data analysis.
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