Encyclopedia of Experiments: Immunology
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Transcript
Use one micromolar of cell-permeant green fluorescent 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 5 minutes to pellet the cells.
Resuspend in phosphate-buffered saline to wash the cells. Then, centrifuge the cells at 300 times g for 5 minutes. Based on the flow rate, suspend 5 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 withdraw mode, with the pump volume at 0 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 slide 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 1-milliliter pipette. Once the tubing is filled, squeeze it and connect it to the flow chamber. Before perfusing the leukocytes, add 3 millimolar of calcium chloride and 2 millimolar of magnesium chloride to the cell suspension. Then, incubate the cells at 37 degrees Celsius for 5 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 deterrent cells between 2 to 6 minutes using a fluorescence microscope with 10 times magnification connected to a digital camera.
