Infinite f50 absorbance microplate reader

Interleukin 8 (IL-8) release was measured using enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) endotoxin at 0.5 ng/mL concentration was used as a pro-inflammatory positive control. LPS was added to TT1 cells at 0.5 ng/mL suspended in 5% newborn calf serum in DCCM-1 medium. Following treatment, the conditioned media were aspirated from the wells. Measurement of IL-8 release was then determined according to the manufacturer’s instructions (Human CXCL-8 Standard TMB ELISA Development Kit, Peprotech Inc., Rocky Hill, NJ, USA). The optical density was measured at 492 nm in an Infinite^® F50 absorbance microplate reader (Tecan, Nänikon, Switzerland).

Cells were seeded in 12-well plates (4 × 10⁵ cells/well). The infection model was established as described previously. Cell culture supernatant was collected for detection according to manufacturer’s protocol (Beyotime Biotechnology). The absorbance was read at 490 nm with Infinite® F50 Absorbance Microplate Reader (Tecan, Switzerland).

Cancer cell lines were seeded in 24-well plates as follow: 22Rv1, LNCaP, LNCaP-LN3, LNCaP-PRO5 at 1 x 10⁵ cells/well; V16D, MR42D, MR49F, CAMA-1, ZR-75-1, MD-MB-231 and MCF-7 at 5 x 10⁴ cells/well; PC-3, DU-145, SW780, SW1710 and RT4 at 8 x 10⁴ cells/well. Twenty-four hours after seeding, adenoviruses were transduced at a multiplicity of infection (MOI) of 2. Seventy-two hours after infection, cells were lysed, and a luciferase assay was performed as described (Promega, Madison, WI, USA). Protein content was estimated by adding 250 μl of Bradford reagent (ThermoFisher Scientific, Waltham, ON, Canada) to 3 μl of total lysate. Absorbance was then read using an Infinite F50 absorbance microplate reader (TECAN, Mannedorf, Switzerland) at 595 nm. SV40-Luc virus was infected in parallel to normalize for infection efficiency between different cell lines. For androgen sensitivity assessment, cells were treated with 10 nM dihydrotestosterone (DHT) (Toronto Research Chemicals, Toronto, ON, CA) and 10 μM Bica (Sigma-Aldrich, St.Louis, MO, USA) in 10% charcoal-treated FBS (FBS-CT) (Wisent Bio Products), 24 h post-infection. Luciferase assays were performed after 48 h of treatment.

22Rv1, LNCaP, LNCaP-Pro5, LNCaP-LN3 MR-49F, MR-42D, LAPC4, PC-3, DU145, and WPMY-1 cells were seeded in 24-well plates. Twenty-four hours later, cells were transduced with indicated adenoviruses at a multiplicity of infection of 5. To assess androgen responsiveness, all of the cells were cultured in their respective medium supplemented with 10% charcoal-stripped FBS with or without the addition of 10 nM DHT. Seventy-two hours after treatment, luciferase assays were performed to measure luciferase activity. Briefly, cells were lysed using a passive lysis buffer (Promega, Madison, WI, USA) and luciferase activity was measured by means of either Luminoskan Ascent (ThermoFisher Scientific, Ottawa, ON, Canada) or TriStar² (Berthold Technologies, Oak Ridge, TN, USA) following the addition of D-luciferin, as stated in the Promega luciferase assay system. Relative luciferase activity (RLU) was normalized to total protein amount in each well (RLU = RLU ÷ μg of protein). Protein content was determined by adding 250 μL of Bradford reagent (ThermoFisher Scientific) to 3 μL of total lysate. Absorbance of the corresponding lysate was then read at 595 nm using an Infinite F50 absorbance microplate reader (TECAN, Mannedorf, Switzerland).

Cells were seeded onto 96-well plates at a density of 30,000 cells per well. The second day after seeding, the cells were counted and pretreated with 20 μM Z-VAD-FMK (Beyotime Biotechnology), 1 μM necrosulfonamide (MCE, Monmouth Junction, NJ, USA), 1 μM GSK’872 (MCE), 10 μM necrostatin-1 (MCE), or DMSO vehicle control for infection or transfection. LDH released was determined by the LDH cytotoxicity assay detection kit (Beyotime Biotechnology). The cell culture supernatant was collected for detection. The absorbance was read at 490 nm with Infinite^® F50 Absorbance Microplate Reader (Tecan, Switzerland). Cell viability was measured based on the intracellular ATP levels using CellTiter-Lumi™ Plus Luminescent Cell Viability Assay Kit (Beyotime Biotechnology). An equal volume of Celltiter-LUMI™ Plus reagent was added to the cell culture medium to induce cell lysis by oscillation. After incubation at room temperature, the luminescence signal was measured by Synergy^TM 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).

PD-1 levels in plasma were evaluated using a human PD-1 ELISA kit (Sino Biological, Beijing, China). The absorbance was assessed by use of a Tecan Infinite F50 Absorbance Microplate Reader (Tecan, Männedorf, Switzerland). Plasma PD-1 levels were quantified using a standard curve.