2.4.4. Neutrophil locomotion and chemotaxis assay

Chemotaxis assay can be carried out using a special self-constructed apparatus called chemotaxis assembly, which consists of an upper chamber containing the neutrophil cells suspension separated by a micropore filter from a lower chamber – which contains the chemotactic factor i.e. isolated compounds. The assembly is a self constructed apparatus using a 5 mL beaker in a sandwich box with holes bored in its lid contains the lower chamber and Sawnoff tuberculin syringe containing the upper chamber with the filter glued to its lower end is inserted through the hole into the lower chamber in the beaker.

About 10⁶ cells/mL neutrophil cells were prepared in Hanks (buffer) solution. The lower chambers of the chemotaxis assembly with appropriate chemotactic agent maintained at a pH 7.2.

Chamber 1: Hank’s solution (Normal control)

Chamber 2: Casein 1 mg/ml (positive control)

Chamber 3: Pre-determined concentrations of the isolated compounds and the fractions – PGA, PGF, FGS and FGT (100, 50, 25, 12.50 and 5.00 μg/mL in separate chemotaxis assemblies).

The upper compartments filled with the neutrophil cells suspension ensuring that the fluid level in the upper and lower chambers was maintained the same to avoid gradient disturbance. The filters were allowed for wetting from the top before putting them into the lower compartments, when the contents of upper compartments were placed in the lower compartments. The concentration of chemotactic factor throughout the filter was zero before its placement in the lower compartment and after placement of the filtered in chemotactic solution, the gradient began to form the bottom of the filter. The arrangement was maintained steady until the end of the experiment. Then it was incubated at 37 °C in air for 3 h. After about 45 min the upper compartments were removed from the assembly and inverted them to empty fluid out of them. The fluid was fixed by immersing the filters in 70% ethanol or methanol. After few minutes immersion in alcohol, the glue melted and the fluid became loose, which was picked off gently using dental packing forceps. It was stained with haematoxylin and xylol respectively, mounted under microscope using glycerin and covered with a coverslip. The cell migration was measured microscopically by counting the number of cells that have reached the lower surface of the filter after a given time interval. The count of lower surface was taken, as the count of cells on the lower surface is directly proportional to the number of cells placed on top of the filter at the start of the experiment (Wilkinson, 1981, Reevas, 1994, Chaning and Rodger, 1994).