An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

For the filter model, first cut a piece of Parafilm to the same size as the glass coverslip, using a metal template. Then, cut out a 20-by-4 millimeter slot in the Parafilm using a metal template to create the flow channel. Use the Parafilm gasket to mark the flow channel on the glass cover slip. Align the edges of the six-well filter on the glass cover slip. Make certain that the filter covers the flow channel markings. Carefully cut out the filter using a type 10A scalpel.

Next, carefully cover the filter with a Parafilm gasket, ensuring that the flow channel slot is in the middle of the filter. Use a piece of clean tissue, and push out any bubbles. Now, place the coverslip in the recess of the bottom plate of the Perspex flow chamber.

Position the top Perspex plate over the top of the gasket, and screw the plates together. Turn the three-way tap to allow the PBS-A wash buffer to flow through the valve. Then, connect the manometer tubing to the inlet port of the top Perspex plate. Run PBS-A through the flow channel to allow bubbles to pass through.

Then connect the manometer tubing from the syringe pump into the outlet port of the Perspex plate. Clean any PBS-A that has dripped onto the upper plate of the chamber. Finally, place the chamber on the stage of an inverted phase-contrast microscope, and adjust the focus to visualize the endothelial cells above the filters.

To perfuse leukocytes over endothelial cells, set the syringe pump to refill, and press Run. Once the focus has been adjusted to visualize the endothelial cell monolayer, put two milliliters of purified neutrophils into the sample reservoir, and leave to warm for two minutes. Then, wash the endothelium with PBS-A for two minutes. Turn the valve on to perfuse neutrophils over endothelium. Deliver the neutrophil bolus for four minutes. Turn the valve off and perfuse PBS-A from the wash reservoir for the remainder of the experiment.

When performing the experiment, ensure that air bubbles do not pass through at any point during the assay, as this will disrupt the endothelial cell monolayer, and cause detachment of adherent neutrophils.

Record neutrophil recruitment either during neutrophil flow or postperfusion using digital image capture software. Identify the center of the channel by moving the objective to the edge of the channel at the inlet port and identifying the middle of the port. Make all digital recordings of at least 5 to 10 fields in the center of the flow channel.

While recording during neutrophil flow, take images of a single field every 10 seconds for one minute. Move along the channel, and record another field for one minute, repeating this process for the duration of the bolus.

For recording postperfusion, make a 10-second recording of 5 to 10 fields down the center of the flow channel for assessing leukocyte behavior. Take images every second within the 10-second interval. This allows sufficient time for capture from flow and the behavior of adherent neutrophils to be analyzed.

Record a single field containing at least 10 transmigrated neutrophils for five minutes, taking images every 30 seconds. This can be used to calculate the velocity of migrated cells. Record another series of 10-second fields. This allows neutrophils time to migrate through the endothelial monolayer.

Finally, stop the syringe pump, and remove the outlet tubing and then the inlet tubing. Disassemble the flow chamber, and rinse the sample reservoir with PBS-A. Repeat for subsequent filters and microslide channels before proceeding to analysis of leukocyte recruitment and behavior as described in the text protocol.