Annexin 5 fitc and pi

Flow cytometry assay was conducted to evaluate cell‐cycle distribution and apoptosis in transfected RCC cells. In brief, cells were seeded in 6‐cm dishes at a density of 2 × 10⁵ cells per well and cultured overnight. The next day, cells were harvested and fixed in 70% ice‐cold ethanol overnight at 4 °C. After being washed with PBS, cells were incubated with propidium iodide (PI) (Sigma‐Aldrich, St. Louis, MO, USA) for 15 min in the dark for cell‐cycle analysis. For apoptotic assay, cells were incubated with Annexin V–FITC and PI (Abcam, Cambridge, MA, USA) in the dark for 15 min. Afterward, stained cells were analyzed for cell‐cycle distribution, or apoptotic rate by the distribution and apoptosis of each phase of the cell cycle were measured by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Propidium iodide (PI) was applied to analyze cell cycle, and Annexin V-FITC and PI (Abcam Co., Cambridge, MA, USA) were used for analysis of cell apoptosis rate. The CaSki cells were washed twice with PBS and added with 0.25 % pancreatin for digestion. After adjusting the density to 10⁵/ml, the cells were incubated with fluorescent antibody at room temperature for 30 min. The cells were precipitated and suspended after 200 × g centrifuging. Finally, the flow cytometer (BD Bioscience, San Jose, CA, USA) was applied for analysis. The experiment was repeated 3 times, and the average values were obtained.

In order to assess the effect of IDO1 on the chondrocytes, 1×10⁶ cells/well were cultured in a 96-well plastic plate. Then 50 ng/ml/well of IDO1 was added at different time points (24, 48, 72, and 96 h) compared to PBS. As well, the same experiment was repeated using Epacadostat 50 ng/ml/well compared to PBS. Cck8 buffer was added according to the instructions of the kit (Yeasen, USA). After 4 h, OD was measured by microplate reader at 450 nm.
To confirm the apoptotic effects of IDO1 on the chondrocytes, annexin-v assay was performed. Briefly, 1×10⁵ cells were cultured with 50 ng/ml IDO1 for 72 h. Later annexin-V-FITC and PI were applied according to the kit instructions (BioVision, Canada). Then, the apoptosis was visualized using confocal laser microscopy.

(MACSQuant, Miltenyi Biotec, Bergisch Gladbach, Germany): For absolute cell number, the cells were harvested in a fixed volume and counted. For annexin-PI assay, cells were harvested and incubated with annexin V-FITC and PI (BioVision Inc., Milpitas, CA, USA) according to manufacturer’s instructions. Annexin−/PI−, surviving cell fraction; annexin +/PI−, early apoptosis; annexin +/PI+, late apoptosis and annexin−/PI+, late apoptosis/necrosis. For cell cycle analysis the cells were stained with PI, harvested and the different cell cycle phases (SubG1, Go/G1 and S-G2M) were gated and analyzed. For αvβ3 estimation, the cells were harvested in RPMI 1640 and labeled with 10 μg/ml PE-αvβ3 antibody (LM609).

MACSQuant (Miltenyi Biotec) was used. For absolute cell number, the cells were harvested 72 hours post trasnfections, in a fixed volume and counted. For annexin-PI assay, cells were harvested 72 hours post trasnfections and incubated with Annexin v-FITC and PI (K101–400, BioVision Inc.) according to manufacturer's instructions. Annexin−/PI−, surviving cell fraction; annexin+/PI−, early apoptosis; and annexin+/PI+, late apoptosis/necrosis. For cell cycle analysis the cells were harvested, 72 hours post trasnfections, permeabilized by 70% ethanol for 20 min at −20°C and stained for 15 min at room temperature with DNA propidium iodide (PI) (50 μg/mL)/RNAse A (10 μ g/mL) (P4170, Sigma-Aldrich).

T24 WT and T24 Mut/T24 Mut2 were cultured with/without 10 μg/ml epirubicin for 24h and 48h, and apoptosis was assessed with the Annexin V-FITC kit according to the manufacturer's instructions. The cells were washed twice with cold PBS, digested, collected, and resuspended to binding buffer. Annexin V-FITC and PI were added (BioVision, Milpitas, CA, USA), and the cells were incubated for 10 min at room temperature in the dark. Then, 200 μl binding buffer was added, and the Annexin V positive cells were analyzed using a FACSCalibur flow cytometry system (BD Biosciences, US).