Japanese cedar pollen

Urban dust collected on filters from the central ventilating system of a building in the centre of Beijing city between 1996 and 2005 (CRM No. 28, National Institute for Environmental Studies, Ibaraki, Japan) and Japanese cedar pollen (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) were used in the present study.

Preparation of the Pollen BG was followed by the procedure used in a previous paper [7 (link)]. To prepare the BGRP column, a Hitrap NHS-activated column (Cytiva, Marlborough, MA, USA) was conjugated with S-BGRP according to the manufacturer’s protocol. Before use for BG purification, the BGRP column was washed with 10 mL of PBS that was flowed through a peristaltic pump set at 2 mL/min. The pollen extract was prepared as follows. Five grams of Japanese cedar pollen (Fujifilm Wako, Osaka, Japan) were suspended in 1 L of 0.1M NaHCO₃ aqueous solution and stirred for 30 min at room temperature. Then the supernatant was collected by 2 step centrifugations with 6000 × g for 10 min and 10,000 × g for 10 min. Finally, the supernatant was filtered with a 0.20 µm aPES bottle top filter (Thermofisher Scientific, Waltham, MA, USA). Then, 900 mL of the pollen extract was passed through the column at a rate of 2 mL/min. After washing the column with 10 mL of PBS (wash fluid), the BG was eluted as five fractions (900 µL/fraction) using 0.03 M NaOH. The eluates containing Pollen BG were immediately neutralized with 300 µL of 0.1 M phosphate citrate buffer (pH 3.0), dialyzed against deionized water, and lyophilized.