The overall goal of this procedure is to demonstrate a method to separate neutrophils from a drop of whole blood and then perform a subsequent chemotaxis assay using a single microfluidic device. This method provides our code to perform a neutrophil chemotaxis assay using small volume of whole blood. And can be easily applied by researchers to answer biological questions related to immune cell migration.
The main advantage of this technique is integration of and our chemotaxis assay on single microfluidic device. This permits assembled and analysis in 25 minutes. To begin, prepare the master maldus described in the accompanying text protocol.
Then silanize the surface of the SU8 mold to facilitate the release of PDMS from the mold. Next, mix 40 grams of PDMS base and four grams of the carrying agent in a plastic beaker. Place the prepared SU8 master mold in a petri dish and carefully pour the mixed PDMS solution onto the mold.
Place the petri dish in a dessicator and apply a vacuum to de-gas the PDMS solution for 20 minutes. Then, place the petri dish in an oven and cure the PDMS at 80 degrees Celsius for two hours. After baking, carefully cut and peel off the PDMS slab from the SU8 mold.
Cut the PDMS slab to separate it into individual devices. Trim the PDMS device to make two walls around the cell loading port for holding the magnets. Next, punch out the cell loading port using a three millimeter diameter puncher.
Then, switch to a six millimeter punch and remove the chemical inlet reservoirs in the waste outlet. Using a piece of adhesive tape, remove the dust on the surface of the PDMS slab. Place the clean slab in a clean glass slide into the plasma machine.
Apply the vacuum for three minutes. And then turn on the plasma power and set the level to high. Gently adjust the air valve and plasma treat the PDMS and the glass slide for three minutes.
When finished, turn off the plasma power and release the vacuum. Carefully take out the PDMS slab in the glass slide using tweezers, immediately place the PDMS slab on top of the glass slide, with channel structures face down gently press the PDMS slab to bond it to the glass and then immediately fill the microfluidic channel with deionized water. The PDMS were generally pressed onto the glass substrate to avoid in the well in barrier channel.
Remove the deionized water from the device and add 100 microliters of Fribronectin solution from the outlet. Wait three minutes to insure 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 ten microliters of whole blood into a 1.5 milliliter tube. Then, add two microliters of the antibody cocktail. And 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 and 100 microliters migration medium to their designated inlet reservoirs using two pipettors. This will generate a chemoattracting gradient by continuous laminar flow based chemical mixing. The chemoattractant can be recombinant proteins such as FMLP or a clinical sample such as the supernatant of sputum from patients with COPD.
Once flow has stabilized, acquire fluorescence images of the FitZ dextron 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 ten times objective to record the cell's final positions for data analysis.
Neutrophils are negatively selected for from a drop of whole blood directly in the microfluidic device using magnetic particle antibody labeling and a pair of magnets. In order to confirm the purity of the on chip isolated neutrophils, Giemsa staining was performed. The results show the typical ring shaped and lobe shaped nuclei of neutrophils.
Following cell loading, the docking structure effectively aligns the cells next to the gradient channel before applying a chemical gradient. When exposed to a chemical gradient for 15 minutes chemotaxis occurs. And can be measured using imaging.
These results show that after 15 minutes, few cells crawled through the barrier channel in the medium controlled sample. By contrast, many neutrophils rapidly moved through the barrier channel and migrated toward a 100 nanomolar FMLP gradient. Once this can mail chemotaxis directly from our in 25 minutes.
This offers a useful tool for researchers to study the mechanics of whole cell migration. And a tool for clinical applications.
