Alphaphot 2 ys2 microscope

The antichemotaxic assay was performed using a 48-well plate, as described by Boyden[8 (link)] with minor modifications introduced by Andrade et al.[9 (link)] The dried ethanolic extracts dissolved in DMSO (final concentration of up to 1%) were assayed at concentrations of 0.01, 0.10, 1.0 and 10.0 μg/mL. Mangiferin was tested at concentrations of 0.1, 1.0, 10.0, 50.0 and 100.0 μM and indometacin (50 μM) was used as the reference drug. All the samples were dissolved in Hanks’ balanced salt solution (HBSS, pH 7.4) to obtain final concentrations for the test.
To obtain the rat polymorphonuclear neutrophils, 20 mL of sterile 1% glycogen (w/v) were injected into the peritoneum of Wistar rats and four hours later, the animals were killed by decapitation and the leukocytes collected. The cell pellets were washed, suspended in HBSS, in order to obtain a leukocyte density of about 1.5 × 10⁶ cells mL^-1 and kept on ice until use. Chemotactic migration of leukocytes was measured using the micrometer on a fine-focus knob of a Nikon Alphaphot-2 YS2 microscope. The distance from the top of the filter to the plane of the farthest focus containing only two cells in ten microscopic fields, allowed leukocyte migration to be determined. Samples were tested in duplicate.