Sterile polypropylene centrifuge tubes

THP1 and A549 human cell lines were obtained from ATCC and cultured in RPMI-1640 + GlutaMAX (Gibco, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco, USA), penicillin, and streptomycin (Gibco, USA). All cell culture experiments were carried out in a humidified incubator at 5% CO₂ and 37 °C. Adherent cells were harvested by trypsin incubation using 0.05% trypsin in EDTA (Gibco, USA). PBMCs were extracted from buffy coats collected from healthy blood donors by the NZ Blood Service under ethical approval from the Northern B Health and Disability Ethics Committee (reference NTY/10/08/065/AM01). Buffy coats were diluted with phosphate-buffered saline (PBS, pH 7.4) and layered over Histopaque 1077 (Sigma-Aldrich, St Louis, MO, USA) in 15 mL conical sterile polypropylene centrifuge tubes (Greiner Bio-One, Frickenhausen, Germany, The cells were centrifuged at 400 g for 30 min, the interphase removed and washed three times in 10 mL of sterile PBS and centrifuged at 250 g to remove residual platelets. Finally, the cells were quantified in a Neubauer chamber (Fortuna, Wertheim, Germany) and viability was assessed by 0.4% w/v trypan blue exclusion (Sigma-Aldrich) in PBS. Samples were used in the experiments if viability exceeded 85%.

Small branches of C. japonica (Cj1, Cj2, and Cj3) were collected from an accessible height of approximately 3 m above the ground on 19 March 2010 using long reach pruning shears while preventing branches from falling on the ground (Fig. 1). The collected branches were placed in clean polyethylene bags without touching with bare hands and brought to the laboratory, after which leaves about 5 cm in length with small stems were separated from the branches without distinction of leaf age and used for extraction of microbial genomic DNA. Leaf samples (10 g) were placed into 50-mL sterile polypropylene centrifuge tubes (Greiner Bio-one, Frickenhausen, Germany), after which 30–40 mL of sterile potassium phosphate buffer (pH 7.0) was added. Microbial cells from the leaves were collected by ultrasonic disintegration (45 kHz, 10 min) and centrifugation (5800×g, 10 min, 5 °C), after which the DNA for genetic analyses was extracted from the precipitates using a FastDNA SPIN kit for Soil (Q-Biogene) according to the manufacturer’s instructions. All sampling and DNA extraction processes were conducted while wearing a clean pair of nitrile rubber gloves.