Macrophage phagocytosis assay

Macrophages were prepared from Kunming mice according to the reported procedure [13]. Macrophages phagocytosis was measured by a neutral red uptake method as described in the reported data [14, 15]. Peritoneal exudate cells were collected from the S-180-bearing mice by lavage of the peritoneal cavity with sterile physiological saline. After centrifugation (370 g, 10 min), the erythrocytes were lysed with Tris-NH₄Cl, and the cells were then washed three times and re-suspended in complete medium at 2 × 10⁶ cells/ml. Cell suspension (100 µl) was added in each well of 96-well microwell plates. After a 3-hour incubation to allow the cells to attach to the plate bottom, the supernatant was discarded, and 0.075% of neutral red dye was added to each well (100 µl per well). The plates were incubated for another 1 to 2 hours. The plates were then washed two times with phosphate buffered saline (PBS) and were patted gently on tissues to let them drain. Finally, 100 µl of lysis solution (0.1 mol/l acetic acid and ethanol in the ratio of 1: 1) was pipetted into each well. The mixtures were blended completely and evaluated at a wavelength of 540 nm on a Bio-Rad microplate reader. Each experiment was performed in triplicate.