Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The overall goal of this experiment is to investigate the mechanisms behind the interactions of leukocytes to adherent vascular cells or platelets under flow conditions. This method can help answer key questions in the immunology field such as how leukocytes are recruited to sites of inflammation. The main advantage of this technique is that it's cost effective, user friendly, and allows the investigation of a great variety of experimental conditions.

This method can provide insight into leukocyte adhesion. It can also be applied to other systems such as the adhesion of tumor cells to immobilized cells or substrates under flow conditions. Demonstrating the procedure will be Annemiek Dickhout, an engineer and a PhD student working in my laboratory.

After obtaining blood samples, add 1/15th volume of acid citrate dextrose to the blood. Next, centrifuge the blood sample by setting the brakes off at 350 times g for 15 minutes to obtain platelet-rich plasma. Then transfer the supernatant obtained to a 15 milliliter conical tube and add 1/10th volume of acid citrate dextrose to it.

Again, centrifuge the suspension by setting the brakes off at 1, 120 times g for 15 minutes to obtain platelet-poor plasma. Next, discard the supernatant. Next, carefully resuspend the platelet pellet in platelet buffer maintained at pH 6.6 using a Pasteur pipette with a wide bore.

Then add 1/10th volume of acid citrate dextrose to it and gently mix it. Next, centrifuge the platelets at 1, 120 times g for 15 minutes to form the pellet. After the centrifugation is over, discard the supernatant and resuspend the platelets in one milliliter of platelet buffer maintained at pH 7.5.

Then use an automated hematology analyzer to measure the platelet concentration in 1/10th dilution. Finally, adjust the cell count with platelet buffer maintained at pH 7.5. To secure the adherent platelets, immerse the glass coverslip in 1.2 molar 50%volume by volume of hydrochloric acid ethanol mixture.

After immersing, wash the coverslip twice with ultrapure water. Then use nitrogen to dry the coverslip. Next, use 100 microliters of 30 micrograms per milliliter of rat collagen type one to pre-coat the glass coverslip.

After coating the glass coverslip, incubate the coverslips at room temperature for 30 minutes. Then expel the collagen solution and wash the coverslip with phosphate buffered saline thrice. Next, use 1%bovine serum albumin in HEPES buffer to block the collagen coated coverslips for 30 minutes at room temperature.

After 30 minutes, wash the coverslips and then dry them to mount in the perfusion chamber. After mounting the coverslips, keep the platelets ready. To immobilize the platelets, transfer 70 microliters of platelet suspension to a channel and incubate for 1-1/2 hours at 37 degrees Celsius and 5%carbon dioxide.

Then replace the nonadherent platelets with blocking buffer for 30 minutes at room temperature. Use one micromolar of cell-permeant green fluorescence nucleic acid stain to fluorescently label the leukocytes. After labeling, incubate the leukocytes for 30 minutes at 37 degrees Celsius.

Then centrifuge the cells at 300 times g for five minutes to pellet the cells. Resuspend in phosphate buffered saline to wash the cells. Then centrifuge the cells at 300 times g for five minutes.

Based on the flow rate, suspend five milliliters of the cells in assay buffer. Then heat a water bath to maintain 37 degrees Celsius. Next, set aside a 50 milliliter perfusion syringe and install it on the syringe holder.

Then set the pump on with draw mode with the pump volume at zero milliliter and diameter at 26.70 millimeter. Next, attach a Luer lock coupler to the syringe and connect the tubing with the elbow Luer connector to the Luer lock coupler. Then attach the free end of the tubing to the flow chamber.

To adhere the leukocytes to the immobilized platelets, connect the microslide with the syringe. To perfuse the leukocytes and then adhere to a bed of vascular or platelet monolayer, attach a second tubing to a 50 milliliter conical tube containing the assay buffer and then fill the tubing using a one milliliter pipette. Once the tubing is filled, squeeze it and connect it to the flow chamber.

Before perfusing the leukocytes, add three millimolar of calcium chloride and two millimolar of magnesium chloride to the cell suspension. Then incubate the cells at 37 degrees Celsius for five minutes. To remove possible air stuck in the chamber and in the tubing, perfuse with assay buffer before perfusing the cells.

Next, squeeze the tubes to close the end and switch the tubing from the assay buffer to the cell suspension thereby preventing any trapped air bubbles within. For the first cells to arrive, perfuse the cells at appropriate flow rate and shear stress. Keep perfusing the cells, maintaining the flow rate and shear stress for two minutes.

Then capture six images of the rolling and adherent cells between two to six minutes using a fluorescence microscope with 10X magnification connected to a digital camera. In this assay, adhesion of green fluorescently labeled THP1 cells with the endothelial cells was analyzed in the presence or absence of tumor necrosis vector alpha stimulation. The graph and the fluorescence images obtained show significant increase in THP1 cell arrest upon stimulating the endothelial cells with tumor necrosis vector alpha in comparison to the control.

When recorded in time lapse, transendothelial migration of primary monocytes can be monitored over time. Next, adhesion of fluorescently labeled neutrophils with immobilized platelet monolayer in a flow chamber was analyzed in the presence or absence of TRAP-6 stimulation. The plot obtained shows a sharp increase in the adhesion of neutrophils to the TRAP-6 stimulated platelets in comparison to the control.

Next, platelets were obtained from JAM-A deficient mice to study its adhesion with raw 264.7 mouse monocyte in the presence of surface receptor inhibitors. The graph and the fluorescence images obtained show significant decrease in the adhesion of the mouse monocytes with the platelets when compared to the control. Further, to study the adhesion of raw 264.7 cells with platelets, JAM-A positive and deficient mice were exposed to incremental increase of shear stress.

From the graph, it's clear that the adhesion of raw 264.7 cells with platelets obtained from JAM-A deficient mice are more resistant to shear stress than JAM-A positive platelets. Once mastered, this technique can be done in one day if performed properly. While attempting this procedure, it is important to very gently wash your platelets during isolation so that your platelets do not become pre-activated or aggregated.

It is also crucial to make sure that you don't have any air bubbles in your flow system because the system might get congested and to ensure a stable and even shear stress. Following this procedure, underlying mechanisms of leukocyte recruitment during vascular inflammation can be investigated.