1.5 ml microtube

ADCP assays were performed using THP-1 cells in suspension and centrifuged during the washing steps at multiplicity of infection (MOI) of 5 bacteria per cell (CFU/cell). A total of 200 μL of B. pseudomallei K96243 at 1× 10⁶ CFU/mL were incubated with 20 μL of diluted antibody in PBS or diluted pooled serum in RPMI (5 μL serum in 45 μL RPMI) in a 1.5-mL microtube (Axygen, Corning, USA) at 37°C for 1 h. The opsonized bacteria were then washed with RPMI twice by centrifugation at 4,000 × g for 15 min. The pellet was resuspended with 220 μL of RPMI, then transferred into 96-well U bottom plate with THP-1 cells and incubated at 37°C with 5% CO₂ for 2 h. Each sample was performed in triplicate (25 (link), 26 (link)).
Opsonized B. pseudomallei K96243 was prepared as described above and incubated with THP-1 cells for 2 h. Extracelllular bacteria were removed by washing with RPMI 3 times. THP-1 cells were treated with 500 μg/mL of kanamycin for 2 h. Live and dead THP1 cells were examined using an inverted light microscope. The cells were washed 3 times with PBS and 1 mL of 0.1% Triton X-100 (Thermo Fisher Scientific, USA) in PBS was added and incubated at RT for 10 min. Cell lysates were diluted in PBS and 10 μL was dropped on LB agar. The plates were incubated at 37°C for 24–48 h to determine colony counts (41 (link)).

Before measuring the mating behavior, pupae were separated from the colony and transferred to a 1.5 mL microtube (Axygen, Union City, CA, USA). Newly emerged adults were separated from the rearing colony and age-graded by days after emergence. The adults were individually provided with a diet bean (0.2 × 0.2 cm) until being used for assay. A mating arena used a cap of 1.5 mL microtube covered with cover glass (18 × 18 mm, Marienfeld, Königshofen, Germany) and included a pair of adults under a stereomicroscope for observation. Each mating behavior was examined for 10 min after the introduction of both males and females under the conditions of 25 ± 1 °C temperature and 60 ± 5% relative humidity under a dark condition with an infrared light (Infrared light 150W, Couyor, China) illumination. Copulation behavior within 10 min in the arena was recorded as a successful mating. Each observation was replicated 10 times with every fresh adult.