Treatment of polystyrene latex beads with human IgG or PLL

Beads were coated with IgG at the time of use. Polystyrene latex beads dyed blue with a mean diameter of 0.8 μm were suspended in water at 0.83% (w/w), and 750 μl of the suspension was centrifuged at 15,000 g for 10 min at 4 °C; the supernatant was subsequently removed. Precipitated beads were resuspended in 1 ml water before they were collected again by centrifugation. The procedure for washing with water was repeated three times. Number of the beads was calculated by equation provided by manufacture, and 3.57 × 10⁹ beads were incubated in 400 µl RPMI1640 medium containing human IgG at 37 °C for 1 h. The supernatant was subsequently removed after centrifugation at 15,000 g for 5 min at 4 °C; the precipitated beads were suspended in 1 ml phosphate buffered saline (PBS) and the beads were collected by centrifugation at 15,000 g for 5 min at 4 °C. This procedure for washing with PBS was performed three times. Beads were resuspended in PBS at 8.93 × 10¹⁰ beads/ml. PLL (300 µg/ml) was used to coat carboxylated polystyrene latex beads dyed blue with a mean diameter of 1.0 μm by covalent coupling using the carbodiimide method according to the manufacturer’s protocol (Polysciences, Inc.). Prepared PLL-coated beads were stored at 4 °C until use.