Sterile phosphate buffered saline pbs

Nine-week-old female BALB/cByJ (Charles River Laboratories, L’Arbresle, Lyon, France) were housed in a controlled temperature and pressure environment. Mice were adapted to new environmental conditions for one week before the beginning of the experimental procedure. The animals were kept in cages with water and food ad libitum, enriched with cardboard houses with cotton squares as nests.
Animals were randomly divided into two groups, including a pristane-induced-lupus (PIL) group (n = 5) that received a single intra-peritoneal injection of 500 µl of sterile pristane oil (2, 6, 10, 14-tetramethylpentadecane, Sigma Aldrich, MO, USA) according to Satoh etal. (16 (link)) and a control (CO) group (n = 5) that received a single intra-peritoneal injection of 500 µl of sterile phosphate buffered saline (PBS, Sigma Aldrich). Blood, stool, and urine samples were collected before PBS/pristane induction (Day 0) and at six months post-induction (M6). The animals were observed weekly for clinical monitoring. At the end of the experiment (M6), all animals were euthanized by lethal overdose of Dolethal^® after an anesthetic protocol (including 90 mg/kg of ketamine^® and 10 mg/kg of xylazine^®).

Before cell seeding, electrospun PLGA fleeces with the two different diameter size were cut into rectangular pieces 10 × 5 mm, sterilized with 70% ethanol (EtOH) in 0.9% NaCl/distilled water (diH₂O) for 10 s followed by washing with sterile phosphate-buffered saline (PBS; Sigma Chemical Co. St. Louis, MO, USA) for 10 min as previously reported by Russo et al. [50 (link)]. The fleeces were then conditioned in cell culture growth medium (GM) composed by Minimum Essential Medium Eagle-α modification (α-MEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Ultra-Glutamine, 1% Amphotericin B, 1% Penicillin/Streptomycin, and incubated at 38 °C with 5% CO₂ for 24 h.

ERM infection was performed using Y. ruckeri (CSF007-82) virulent isolate that was obtained from the National Centre for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, USA, and stored at −80 °C in the Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna. Prior use, one colony was inoculated in tryptic soy (TS) broth (Sigma-Aldrich, Austria) and was cultured overnight at 22 °C in an orbital incubator, 144 revolutions per minute (rpm) [31 (link)]. For the experimental challenge, the bacterial suspension was adjusted at a theoretical concentration of 2 × 10⁹ CFU ml⁻¹, which was assessed using a spectrophotometer (Eppendorf BioPhotometer^®, Eppendorf, Hamburg, Germany). Water level was lowered to reach 50 L/tank, then the bacterial challenge was performed by 2 h bath exposure to the bacterial suspension to reach a final concentration of 2 × 10⁶ CFU ml⁻¹ [77 (link)]. Pathogen-free group was exposed to sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, Austria). For spreading the infection, the water flow was temporarily stopped while the airflow was constantly maintained. Then, the water flow was resumed after 2 h of exposure.

Five serovars of Salmonella (Stanley BYC12, Indiana HZC10, Typhimurium YXC1, Thompson LWC10, Kentucky CBC2) isolated from poultry slaughterhouses (Guangzhou, Guangdong, China) in China were used in this study. The bacterial strains were maintained in brain heart infusion broth (BHI, Becton Dickinson (BD), Franklin Lakes, NJ, USA) containing 20% (v/v) glycerol at −80 °C. Each strain was separately incubated in BHI at 37 °C for 24 h and cultured to approximately 9 log CFU/mL. Equal volumes of each culture suspension were mixed to obtain a five-strain mixture of Salmonella. Appropriate 10-fold dilutions in sterile phosphate buffered saline (PBS, Sigma, St. Louis, MO, USA) were made and plated on xylose lysine Tergitol 4 (XLT4, BD) agar to determine the cell number in the inoculums.

Media for each cell line was purchased from Corning Life Sciences (Tewksbury, MA, USA). Media supplements were obtained from Sigma-Aldrich (St. Louis, MO, USA), including penicillin/streptomycin, glucose, glutamine, sodium bicarbonate. FBS, triple-0.1-micrometer-filtered, was purchased from Atlanta Biologicals through R&D Systems (Minneapolis, MN, USA). NiCl₂, Ni-acetate, sterile dimethylsulfoxide (DMSO). Sterile phosphate-buffered saline (PBS) and molecular-grade water were purchased from Sigma-Aldrich (St. Louis, MO, USA), Fisher Scientific (Houston, TX, USA), and Pierce Biotechnology (Rockford, IL, USA). ELISA kits were obtained from Pierce Biotechnology (Rockford, IL, USA), while antibodies were purchased from R&D Systems Inc., (Minneapolis, MN, USA), Cell Signaling Technology, Inc. (Danvers, MA, USA), and Thermo Fisher Scientific (Waltham, MA, USA).

Four ml of chloroform was added to 5 ml of P. aeruginosa culture supernatants prepared as described above. After vigorous vortexing, solutions were let to rest until two distinct phases were apparent. The organic phase contains the pyocyanin and is usually colored (blue-green). The top aqueous phase was discarded without disrupting the organic phase. Pyocyanin was then extracted by adding 1 ml HCl 0.2 M combined to another cycle of vortex and resting of the mixture. The pyocyanin-containing phase then appeared in shades of pink and was retrieved. Aliquots of 200 µl were serially diluted in 96-well plates with sterile phosphate-buffered saline (PBS, Sigma) and absorbance was read at 520 nm, using an Epoch plate reader (BioTek Instruments, Winooski, USA).