For this method can help answer key question in the field of inflammation and immunology. For example, what are the molecular mechanism of leukocyte adhesion and trans-endothelial migration. The main advantage of this technique is that it allows unlimited use discrimination between transmigration and strong addition of monocytes to endothelial monolayer.
Although this method provides information about the recruitment of monocytes on the flow, it can also be adapted to other cells of the hematopoietic systems such as T-cells, B-cells, or derivatives of subpopulation thereof. After washing, label the human umbilical vein endothelial cells or HUVEC with 30 microliters of 37 degree Celsius M199 medium supplemented with one micromolar CMFDA for 10 minutes at 37 degree Celsius and 5%Carbon dioxide. At the end of the incubation, wash the cells with 150 microliters of complete M199 medium and incubate the culture in 150 microliters of fresh complete M199 medium for another 30 minutes.
Then replace the supernatant with 150 microliters of complete M199 medium containing the appropriate experimental supplements and return the slide chamber culture to the incubator for six hours. For human PAN monocyte isolation, dilute a freshly collected blood sample in PBS supplemented with one millimolar EDTA at a one to one ratio and carefully layer 20 ml of the blood solution onto 20 ml of density gradient medium for density gradient separation by centrifugation. Collect the middle peripheral blood mononuclear cell platelet layer into a new 50 ml tube containing 40 ml of PBS supplemented with 1 millimolar EDTA and bring the final volume to 50 ml with additional PBS-EDTA After counting, isolate the monocytes with the PAN monocyte isolation kit according to the manufacturer's instruction.
And wash the cells three times in M199 medium supplemented with 0.5%Bovine Serum Albumin, or BSA, to eliminate any traces of EDTA. The quality of monocyte isolation is critical for ensuring robust transmigration and adhesion result. Thus it is important to strictly follow the manufacturer's recommendation for the isolation.
Resuspend the pellet at a 6 times 10 to the 6 monocytes per ml concentration in fresh M199 medium supplemented with 0.5%BSA and divide the cells into one 200 microliter aliquot per microcentrifuge tube per recruitment assay. Then place the cells at 37 degree Celsius for 20 minutes before their injection into the flow chamber. To prepare the fluidic system for the analysis, first insert a male luer connector into one end of an 8 cm long 3 mm thick piece of silicone tubing and connect the other end to an inline luer injection set.
Then connect the luer connector to one end of a 40 cm long 3 mm thick piece of silicone tubing. Next, the 20 mm syringe to one end of a 1 m long 3mm thick piece of silicone tubing and insert a male luer connector to the other end. Then insert both male luer connectors into a female luer lock coupler and place the free end of the silicone tubing into a reservoir of 37 degree Celsius M199 medium supplemented with 0.5%BSA.
Retract the plunger to fill the tubing with flow buffer and secure the syringe on the syringe pump. Then set the pump to the withdraw mode and specify the flow rate. To connect the slide chamber, clamp the silicone tubing around the female luer lock coupler and disconnect the two luer connector males from the coupler to allow them to be connected to the reservoir of the slide.
Air bubbles not only disturb the image quality but also induce capillary sheer stress to the cells, leading to cell death. To avoid air bubbles, confirm the correct clamping of the tubing before inserting the luer into the reservoir. Then remove the clamps to confirm that the connection is not leaking and place the slide under the microscope.
For imaging of the monocyte recruitment under flow, add 5 microliters of flourescence conjugated anti CD16 antibody and an appropriate nuclear dye to each aliquoted monocytes for a 10 minute incubation at room temperature and collect the cells with a brief centrifugation. Resuspend the pellet in 250 microliters of flow buffer and start the syringe pump. Add 30 microliters of one monocyte suspension to one chamber of the slide and select the 40X objective on the confocal microscope.
Activate the appropriate fluorescent lasers and use the chamber with the labeled monocyte to set the acquisition parameters of the confocal microscope Next, select three fields of view within a half centimeter radius for multi position confocal imaging, and set a Z stack to the 10 to 12 micrometer range and the time lapse acquisition to run every one minute. After three minutes of imaging, inject 200 microliters of monocytes through the inline luer injection port and begin imaging the cellular interaction. After at least 30 minutes, stop the imaging and the flow and clamp the tubing to allow it to be disconnected from the slide.
To determine the transmigration rate of the cells through the stimulated HUVECs, open image J and count the total number of adherent monocytes in each field to determine the cell count per square millimeter. Then count the number of transmigrated monocytes that are present in the basal plane underneath the endothelial cells as identified by the presence of a black hole zone around the nucleus. A simple way to check the activation status of the HUVECs after stimulation is to visualization of their elongation under inflammatory stress.
Time lapse confocal imaging allows visualization of the monocytes at the apical plane where their phenotype can be assessed. Migrating cells undergoing transmigration move to the intercellular cell-to-cell junction before they disappear from the apical plane and reappear in the basal plane. Quantitation of the monocyte recruitment over time confirms that monocyte adhesion is followed by transmigration.
The transmigration rate of CD16 positive monocyte is low when the endothelial cell are stimulated with TNF alpha only but increases when the endothelial cells are stimulated with both TNF alpha and vascular endothelial growth factor or VEGFA. HUVEC stimulation with TNF alpha and TNF alpha plus VEGFA induces an increase in the adherence to CD14 negative T, B and NK cells, compare to CD14 positive monocytes. In addition, HUVEC stimulation induces transmigration of the monocyte population only, as T, B and NK cells do not transmigrate under either TNF alpha or TNF alpha plus VEGFA stimulatory conditions.
To perform an optimal monocyte recruitment assay, it is important that you plan your experiment in advance and do it at the same day. And when you purify the cells, make sure that your microscope is at 37 degrees so that you do not transfer the cells to different temperature during the experiment. To learn this procedure, all the methods like the profiling of HUVEC cytokine and chemokine expression as well as chemotaxis studies can be performed to answer additional question about the molecular mechanism that sustain the transmigration and adhesion of specific monocyte population.
