Chemotaxis of radiolabeled equine neutrophils.

This research article explains a successful method of isolating high purity equine neutrophils, a type of white blood cell, which were then radiolabeled and their chemotaxis (movement) examined in response to different substances. The optimal conditions and factors inhibiting their movement were also studied.

Isolation and Radiolabeling of Equine Neutrophils

• The researchers developed a method to isolate equine neutrophils using metrizamide cushions. This approach resulted in an isolation of cells with a purity greater than 95% and a viability greater than 90%.
• The isolated neutrophils were then radiolabeled, which is a process during which a radioactive isotope is attached to the cells. This enables the tracking and visualisation of these cells under a microscope or similar device.

Chemotaxis Examination

• The researchers measured the movement of these radiolabeled neutrophils in response to various substances using Boyden chambers. These substances were zymosan-activated equine serum, the partially purified equine complement component C5a, and a tripeptide named formyl-L-methionyl-L-leucyl-L-phenylalanine.
• The methodology involved placing the neutrophils in one side of the chamber and the substance on the other. Then, the movement of the radiolabeled neutrophils towards the substance was observed, indicating the chemotactic response of these cells to the substance.

Time and Ionic Requirements for Chemotaxis

• The research evaluated the conditions under which the maximum chemotactic response occur. They concluded that maximum movement was observed after 2 hours of incubation time in the presence of 1.0 mM Calcium and 0.5 mM Magnesium.
• The study noted that the substance dinitrophenol, within a concentration range of 10(-2) to 10(-6) M, inhibited the movement of these cells, thus indicating an antagonistic effect on the chemotaxis of equine neutrophils.

Checkerboard Assay Results

• The researchers employed a Zigmond-Hirsch checkerboard assay, a technique used to distinguish between chemotactic and chemokinetic effects.
• They found pronounced chemotactic activity or cellular movement of the neutrophils towards both zymosan-activated equine serum and the partially purified equine complement component C5a.
• However, the neutrophils did not respond to the formyl-L-methionyl-L-leucyl-L-phenylalanine, implying it doesn’t excite chemotaxis in equine neutrophils.