Sumilon

Clonogenic survival assay was performed as described previously [39 (link)]. In brief, the appropriate plating density was aimed at producing 20–40 surviving colonies in each T-25 flask. After incubation for 14 days, the colonies were fixed and stained with 0.3% methylene blue in ethanol, and colonies containing more than 50 cells were counted as survivor. At least three parallel samples were scored in three to five repetitions performed for each irradiation condition. Clonogenicity and spheroid formation ability assays for CD44+/ESA+, CD44+/CD24+ and CD44−/ESA−, CD44−/CD24− cells sorted from PK45, PNAC1, MIAPaCa-2 and BxPc-3 cells plated in triplicate in a 6-cm dish or a 96 well spheroid formation plate (Sumilon, Sumitomo Bakelite Co., Ltd, Tokyo, Japan) were performed as described previously [40 (link)]. The data is presented as percentage of the wells that contained spheres. and the average size using WinRoof 5.6 software (Mitani Corporation, Tokyo, Japan) after 1-week incubation.

The collected blood was immediately centrifuged at 3000× g for 10 min at 4 °C, and the serum was transferred to a 1.2 mL polypropylene serum tube (SUMILON, Sumitomo Bakelite Co., LTD., Tokyo, Japan). Samples were stored at −80 °C until metabolomic analyses. Frozen samples were thawed and 40 µL aliquots of serum were mixed with 360 µL of methanol containing internal standards (20 µM each of methionine sulfone and camphor 10-sulfonic acid). The samples were vortex-mixed, and 400 µL of chloroform and 160 µL of Milli-Q water (Millipore, Billerica, MA, USA) were added with the mixer, followed by centrifugation at 10,000× g for 3 min at 4°C. MicroMixer E-36 (Taitec Co., Saitama, Japan) was used for vortex. The upper aqueous layer of each sample was filtered through a centrifugal filter tube with a 5-kDa cutoff (Millipore) at 9100× g for 2 hours at 4 °C to remove large molecules. The filtrates were concentrated by centrifugation for about 5 h at 40 °C using Acid-Resistant CentriVap Benchtop Centrifugal Vacuum Concentrator (Labconco Corp., Kansas, MO, USA) and resuspended in 40 µL of Milli-Q water containing other internal standards (3-aminopyrrolidine and trimesate) prior to CE-time-of-flight-MS analysis. The internal standards, 3-aminopyrrolidine and trimesate, were used for only correction migration time.

For preparation of inoculum, tested isolates were cultured anaerobically at 37 °C for 20 to 24 h with Mueller-Hinton broth (MHB; Difco Becton Dickinson and company, Sparks, MD, USA) containing 0.001% pyridoxal hydrochloride (Wako) and the bacterial cell suspensions were adjusted to yield about 5 × 10⁵ CFU/mL. MICs for the Abiotrophia and Granulicatella strains were determined using the microdilution broth method with MHB containing 2.5% lysed horse blood (Strepto hemo supplement ‘Eiken’, Eiken Chemical Co., Ltd., Tokyo, Japan) and 0.001% pyridoxal hydrochloride, according to the consensus guideline from the CLSI for fastidious bacteria [12 (link)]. Briefly, the antimicrobial agents (100 µL/well) were diluted on 96-well round bottom plates (Sumilon, Sumitomo Bakelite Co., Ltd., Tokyo, Japan) in serial two-fold with the supplemented MHB, and 5 µL of the bacterial inoculum was added to each well. The plates were incubated at 35 °C in anaerobic condition for 20 h. The MIC values were defined as the lowest concentrations of antimicrobial agents that completely inhibited the bacterial growth in the microdilution wells, detected by unaided eyes. The strain of S. pneumoniae ATCC 49619 was used for quality control testing, and all MIC values for the strain were within the acceptable limits.