Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

The overall goal of this procedure is to follow the distribution of endogenous junctional, terin, and pcam one during leukocyte trans endothelial migration under physiological flow conditions. This is accomplished by first isolating polymorphonuclear leukocytes or PMNs from the whole blood of healthy volunteers using a proco gradient. The second step is to add fluorescently labeled antibodies to A TNF alpha stimulated human umbilical vein endothelial cell monolayer cultured in flow slides 30 minutes prior to starting the flow experiment.

This will enable visualization of junctional dynamics of endothelial cells during neutrophil transmigration under physiological flow conditions. Next, the flow slide containing the fluorescently labeled TNF alpha stimulated human umbilical vein endothelial cells is connected to the flow system and placed on the microscope stage. The pump will pull the flow buffer from the reservoir through the flow chamber into the syringe.

The final step is to inject the PMNs slowly into the flow system via the injection port. Ultimately, the PMNs are visualized using a confocal laser scanning microscope. After a few minutes, leukocytes appear, adhere and transmigrate.

The experiment can be stopped at any desired moment. The main advantage of this technique over other existing methods like static transmigration assays, is that with this technique you can study adhesion and transmigration in real life under physiological flow conditions. This will help us understand why leukocytes choose one root over the other, so PGA versus transcellular migration.

We do this by visualizing the cell cell junctions with fluorescently labeled antibodies. The human umbilical vein endothelial cells or HU X for this experiment are cultured according to the manufacturer's instructions on fibronectin coated dishes using media supplemented with endothelial growth.Medium. The cell culture is used between four to eight passages when cells reach 80 to 90%Confluence carefully wash them with room temperature PBS at pH 7.4 and then trypsin is the cells after trypsin and centrifugation.

We suspend at 800, 000 cells per milliliter using media. The experiment uses flow chambers that have been coated with fibronectin on the previous day plate. 80, 000 cells in every single channel of the fibronectin encoded flow chambers and gently pipette the cell suspension up and down culture overnight in an incubator at 37 degrees Celsius and 5%carbon dioxide on the following day.

Refresh the media in the flow chamber slide by gently tilting the slide at a 45 degree angle. It is recommended to only remove the media in the reservoirs and not in the channel itself. Removal of the media in the channels may result in endothelial cell loss and death due to the dragging force of the media caused by pipetting.

Check by phase contrast microscopy If the endothelial cells have formed a monolayer. If the cells are not 100%cofluent, change the media two times a day until they reach 100%confluence. Once the cells have reached 100%co fluency stimulate the cells with media containing the inflammatory mediator.

TNF alpha stimulating uix overnight with TNF alpha results in an inflammatory phenotype of the endothelium IE upregulation of cell adhesion molecules such as ICAM one and VA M1.Prior to isolating polymorphonuclear leukocytes or PMNs. Prepare a flow buffer for washing isolated PMNs and for use in the flow assay to 100 milliliters of a previously prepared stock solution. At 100 microliters of fresh one molar calcium chloride, 2.5 milliliters of human albumin from a 200 grams per liter stock concentration and 0.1 grams of glucose.

Filter the flow buffer using a 0.45 micron filter. A 10%trisodium citrate or TNC solution in PBS at pH 7.4 is also prepared prior to isolation of PMNs. PMNs will be isolated from 20 milliliters of whole blood collected in a sodium heparin ette from a healthy volunteer.

First dilute the whole blood one to one with 10%PBST NNC in a 15 milliliter tube. Next, use a pipette boy at the slowest setting to carefully pipette 20 milliliters of diluted blood carefully onto 12.5 milliliters of per co colloidal solution in water with a density of 1.130 grams per milliliter in a new 50 milliliter tube. The per coll containing tube should be tilted at a 45 degree angle during the addition of the blood.

Carefully placed the tubes in the centrifuge and spin at room temperature for 20 minutes at 800 Gs with low acceleration and with the brakes disabled when centrifugation is complete. Remove all the liquid and fill each tube with ice cold erythrocyte lysis buffer to lye the erythrocytes. Leave the tubes on ice, occasionally inverting them until the suspension turns dark red centrifuge at 500 GS for five minutes at four degrees Celsius with the brakes enabled.

The resulting pellet fraction contains the PMNs Together with the erythrocytes, remove the supernatant and wash the pellet twice in ice. Cold lysis buffer at 500 Gs for five minutes at four degrees celsius. After removal of the supernatant from the second wash, re suspend the pellet with flow buffer at room temperature and determine the PMNs concentration using an automated cell counter.

Lastly, suspend the PMNs in flow buffer at one times 10 to the six cells per milliliter and keep at room temperature to label endothelial junctional VA cadherin and pcam one. Add the pcam LOR 6 47 antibody at a one to 100 dilution and the calcium independent FE cadherin ZI antibody at a one to 50 dilution to VE culture, medium in the flow chamber and incubate at 37 degrees Celsius for 30 minutes. To prepare the PMNs for the assay, place them into a water bath for 15 minutes at 37 degrees Celsius prior to injecting them into the flow system.

Connect tubing to an empty flow chamber and fill with warm flow buffer to prevent formation of air bubbles. When setting up the flow system. Using silicone tubing, connect one side of the empty flow chamber to the syringe pump flow system containing a 20 milliliter syringe.

Place the flow chamber on the microscope stage. The assay will be performed using a confocal laser scanning microscope equipped with the 63 x oil objective and a climate chamber set at 37 degrees Celsius and 5%carbon dioxide. Connect the other side of the empty flow chamber to the reservoir flask filled with 37 degrees Celsius flow buffer and start the syringe pump in order to fill all the tubing with flow buffer, the pump will pull the flow buffer from the reservoir through the flow chamber into the syringe.

This tubing also contains an inline lure injection port, which allows PMNs to be injected with a needle into a running experiment without stopping the flow and creating air bubbles. Next, replace the empty flow chamber with the flow chamber containing TNF alpha treated VE pinch off the tubes before disconnecting and reconnecting them to the chamber containing the VE.To avoid air bubbles in the system, it is critical to pinch and clamp off the tubings. When connecting the slide containing the cells.

Place the flow chamber on the microscope stage, adjust the flow speed to one dime centimeter squared in accordance with the physiological flow speed in post capillary es, which is one to five dines per centimeter squared record differential interference contrast or DIC fite LOR 6 47. Simultaneously using a one milliliter syringe, inject the PMN slowly into the flow system via the inline lure injection port. After a few minutes, leukocytes appear, adhere and transmigrate stop the experiment at any desired moment.

By disconnecting the tubing from the flow chamber and pipetting aff fixative into the flow chamber, the resistance of endothelial monolayers was measured to test whether the antibodies interfered with the barrier function of the endothelium. No change in resistance was observed when either the VE could hear an antibody clone 55 7 H one or the isotype control was added to the cells. In contrast, the VE cod hear blocking antibody CL 75 reduced the resistance dramatically.

RAP analysis revealed no change in fluorescence recovery in the presence or absence of the VE CADHERIN 55 7 H one antibody, indicating that the antibody did not alter the dynamics of VE cadherin, VE cadherin and pcam one distribution during neutrophil trans endothelial migration or TEM in real time was analyzed. The top panel shows a neutrophil outlined in white adhering on the endothelium and crossing the cell to cell junction without harming the distribution of VE coherent and pcam one. During diapedesis, a local dispersion of VE coherent and P chem one can be observed when a neutrophil protrudes through the cell to cell junctions outlined in white is a neutrophil on top of the endothelium.

The yellow outline shows neutrophil membrane that is already underneath the endothelium. After completion of diapedesis, the junctions close and ve coherent and pcam one are relocated at the sites of diapedesis. The borders of the trans migrated neutrophil are outlined in yellow.

Following this procedure, leukocyte trans endothelial migration can be performed in order to answer additional questions such as whether leukocytes cross the endothelium by using the bagga or the transcellular root.