Rpmi 1640 without phenol red

The effects of TiO₂ nanotubes on the cell adhesion (after 24 h) and proliferation (after 72 h) were assessed using the MTT assay. The cells at a density of 1 × 10⁴ cells/well (in 1 ml of complete RPMI 1640 medium) were incubated with sterilized biomaterials in a 24-well culture plates for 24 and 72 h. After the respective co-incubation time, biomaterial samples were washed with phosphate buffered saline (PBS) three times and transferred to a new 24-well plate. Then, 500 μl of culture medium (RPMI 1640 without phenol red; Sigma-Aldrich, Germany) and 500 μl of MTT (5 mg/ml in PBS prepared and filtered immediately before use) were added to each well. After 3 h of incubation at 37 °C in 5 % CO₂, the medium was replaced with 500 μl of dimethyl sulfoxide (100 % v/v; Sigma-Aldrich, Germany). The plate was shaken for 10 min, and then the solution in each well was transferred to a 96-well ELISA plate. The absorbance of the solution was measured at a wavelength of 570 nm with the subtraction of the 630 nm background using a plate reader (Synergy HT; BioTek, USA). The blank groups (TNT incubated without cells) were treated with the same procedures as experimental groups. Culture medium without the TNT served as a negative control in each experiment. All measurements were done in duplicate in three independent experiments.

The strain of P. aeruginosa PAO1, subline Lausanne (PAO1-L), was used throughout this study. PAO1 is a serogroup O2/O5, type b flagellated strain [33 (link),34 (link)] exhibiting moderate virulence [35 (link)]. The genome of PAO1-L has been fully sequenced and found closest to Holloway’s original isolate (data not shown) [36 (link),37 (link)]. Mid-log phase cultures of PAO1-L (3 hour, h) in Luria-Bertani (LB) broth were washed with PBS without Ca²⁺ or Mg²⁺ (Sigma-Aldrich), resuspended in RPMI-1640 without phenol red (Sigma-Aldrich) such that cultures were 20x concentrated, and lysed using a French Press. Lysates were centrifuged to remove debris, filtered through a 0.2 μm filter, aliquoted, snap frozen on dry ice, and stored at -80°C until used. Total protein content in lysates was quantified using the bicinchoninic acid assay (Thermo Scientific).

Neutrophils were isolated freshly from blood taken from 15 healthy volunteers. Venous blood was taken with a butterfly needle into EDTA-tubes (S-Monovette 9 mL, Sarstedt, Germany) and directly used for density gradient centrifugation. 6 mL blood was carefully layered on 6 mL of Lympholyte poly cell separation medium (Cedarlane, Burlington, Ontario, Canada). Samples were centrifuged for 35 min at 500 g without break at room temperature. The plasma and peripheral blood mononuclear cell (PBMC) layers were discarded and the polymorphonuclear cell (PMN) layer carefully taken with a pipette and transferred to a 15 mL tube. PMN layer was washed twice with PBS (12 mL, centrifugation at 450 g, 10 min, room temperature without break) and taken up in RPMI medium (RPMI-1640 without phenol red, Sigma-Aldrich, Munich, Germany). Cells were counted by Trypan Blue exclusion method in a Neubauer counting chamber, without counting of residual erythrocytes. Cells were prepared to a density of 1x10⁶ cells/mL.

All reagents and buffers were at room temperature before starting the PKH-67 labeling step. PKH 67 (Sigma/Aldrich Chemical Company, St. Louis, MO, USA) was diluted according to the manufacturer’s kit directions. PBMCs (3 × 10⁶) were incubated for 48 h under Th17 polarizing conditions as described previously, and then were washed in Dulbecco’s PBS (Gibco/BRL, Grand Island, NY, USA), and were re-suspended in 1 mL of solution C from the kit. The PKH-67 was diluted to 4 × 10^− 6 M in 1 mL of solution C. The cells were combined with dye, and the tube was inverted several times over 3 mins. About 2 mL of FCS was added to the tube, and it was inverted continuously for 1 min. The cells were then transferred to a 15-mL conical tube with 4 mL of RPMI 1640 without Phenol Red (Sigma/Aldrich Chemical Company, St. Louis, MO, USA), and with 10% FCS (BioWhittaker, Walkersville, MD, USA) and washed three times in the same medium. To examine the immunosuppressive effects of Resv, PKH-67 labeled CD4⁺ T cells were co-cultured with HRPTEpiCs at a ratio of 150,000:20,000 [24 (link)] with/without Resv (25, 50 uM) or Tac (1, 10 ng/mL). After 3 days, the harvested cells were examined for proliferation using a FACS Caliber flow cytometer (BD Biosciences, San Diego, CA, USA).

Synovial tissue samples from patients with RA and OA were obtained immediately after opening the knee joint capsule. Synovial tissue pieces up to 9 cm² were excised. Part of the tissue was minced and treated with Liberase TM (#05401127001, Roche Diagnostics, Mannheim, Germany) at 37 °C for 1 h on a shaking platform. The resulting suspension was filtered (70 μm) and centrifuged at 1600 rpm for 10 min. The pellet was then treated with erythrocyte lysis buffer (20.7 g NH₄Cl, 1.97 g NH₄HCO₃, 0.09 g EDTA and 1 L H₂O) for 8 min and recentrifuged at 1600 rpm for 10 min. The pellet was resuspended in RPMI-1640 (Sigma Aldrich, St. Louis, MO, USA) with 10% fetal calf serum (FCS). After overnight incubation, cells were supplemented with fresh medium. The culture medium used was RPMI-1640 without phenol red (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FCS, 4 mM l-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10 mM HEPES (Sigma Aldrich, St. Louis, MO, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma Aldrich, St. Louis, MO, USA), and 10 μg/ml ciprofloxacin (Fresenius Kabi, Bad Homburg, Germany). Passage 4–8 SF were used for experiments. The remaining part of the collected tissue was used for superfusion and immunohistochemistry experiments.