Pbs phosphate buffered saline

Curcumin (Biomol, Hamburg, Germany) was stored at −20 °C and diluted prior to use in cell culture medium to a final concentration of 0.2 µg/mL (0.54 µM). 4 µg/mL curcumin was used to provide high quality images for confocal microscopy and optimum fluorescence detection by FACS analysis. Cells were treated with curcumin for 1 h and then exposed to visible light for 5 min with 5500 lx (curcumin^Light; 10 × 40 W lamps, distance 45 cm, emission spectrum: 400–550 nm) using a Waldmann UV 801AL system (Waldmann, Villingen-Schwenningen, Germany) [18 (link)]. To prevent bias effects by the phenol red containing RPMI 1640 based cell culture medium, tumor cells were transferred to phenol red free PBS (phosphate-buffered saline) (Sigma-Aldrich) during light exposure. Thereafter, PBS was replaced by RPMI 1640 and supplements. Control cell cultures received PBS for 5 min without light exposure. To evaluate the effects of low dosed curcumin and light alone, two respective additional controls were employed; tumor cells exposed to light but not to curcumin, and tumor cells exposed to curcumin but no light. Following light exposure (including all controls), tumor cells were allowed to recover in complete cell culture medium for 24 h before starting adhesion and migration experiments.

We collected RTB gallery tissues from naturally infested P. tabuliformis trees at Tunlanchuan Forest Station (37° 48′ N, 111° 44′ E; average elevation 1400 m; P. tabuliformis 3278 ha) in Shanxi Province. Gallery samples (ca. 0.5 g in fresh weight) were first homogenized into fine pieces by sterile forceps and scissors and then macerated in 0.5 ml 10% PBS (phosphate-buffered saline) (Sigma) to obtain crude extracts containing microbial cells (gallery microbiota).
Second to third instar RTB larvae were randomly collected from beetle galleries of infested host pines at Tunlanchuan Forest Station. Beetles were surface-sterilized with bleach, ethanol, and distilled water [10:10:80 (vol:vol)], kept in a climate-controlled incubator (25 ± 1 °C, RH = 70%, darkness) and starved for 24 h before use.

Milk samples were thawed by placing them at 4 °C the night prior to DNA extractions. The samples were homogenised by inverting the tubes and centrifuged at 4000× g for 30 min at 4 °C. The fatty/cream layer that accumulated at the top was discarded using sterile cotton swabs and the supernatant was discarded. DNA was extracted from the pellet according to the manufacturer’s instructions using the PowerFood microbial DNA Isolation Kit (MoBIO Laboratories Inc., Carlsbad, CA, USA) with modifications as follows: Briefly, the cell pellets were washed with PBS (phosphate-buffered saline) (Sigma Aldrich, St. Louis, MO, USA) and centrifuged at 13,000× g for one minute at 20 °C to discard the supernatants. This process was repeated until the supernatant was no longer cloudy. The pellet was then re-suspended in PBS and treated with 90 µL of 50 mg/mL lysozyme (Sigma Aldrich, lysozyme activity: ≥40,000 units/mg protein) and 50 µL of 5 KU/mL mutanolysin (Sigma Aldrich) followed by 15 min incubation at 55 °C with vortexing at intervals of 5 min. Further, samples were treated with Proteinase k (Qiagen, UK, 28 µL of 20 mg/mL, >600 mAU/mL), incubated at 55 °C for 15 min and then treated according to the manufacturer’s instructions using the PowerFood microbial DNA Isolation Kit protocol. The extracted DNA was stored at −30 °C until further use.