Measurement of Mitochondrial ROS and Mitochondrial Membrane Potential (ΔΨM)

Mitochondrial ROS was measured using MitoSOX Red dye (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008). Briefly, OA chondrocytes were stained with 5.0 μM of MitoSOX Red for 10 minutes at 37°C followed by IL-1 β treatment (5ng/ml). For autophagy inhibition studies and its effect on ROS levels, OA chondrocytes were first treated with inhibitor of autophagy for 2 hours followed by MitoSOX Red staining and IL-1β treatment as above. Loss of ΔΨM was assessed using the mitochondrial-specific fluorescent probe JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide) (Invitrogen, T3168). JC-1 forms multimers in cells with a high red fluorescence indicating normal ΔΨM. Loss of the ΔΨM results in a reduction in the red fluorescence with a concurrent gain in green fluorescence as the dye shifts from multimeric form to monomeric state. Ratio of red to green fluorescence was used as an indicator of loss of ΔΨM. OA chondrocytes treated as above were loaded with JC-1 (5 μM) for 20 minutes at 37 °C, washed with PBS and analyzed by flow cytometry using BD Accuri C6 Flowcytometer. Flow cytometry data was analyzed by Flowjo software (Tree Star Inc. Ashland, OR, USA).

OA chondrocytes were cultured with or without IL-1β for 48 hours. At the end of the treatment chondrocytes were washed with 1x-PBS and incubated with 1 ml staining solution containing 0.5 μg/ml PI, 0.1% sodium citrate, and 0.1% Triton X-100 overnight at 4°C. A total of 20,000 cells were acquired in BD Accuri C6 Flowcytometer and percent apoptotic cells were determined by analyzing sub-G1 population (<2 n DNA content) using FlowJo software [26]. TUNEL assay was performed using a kit according to the manufacturer’s protocols (Biotool, USA #{"type":"entrez-nucleotide","attrs":{"text":"B31112","term_id":"2530481","term_text":"B31112"}}B31112). Nuclei were stained with DAPI and apoptotic cells were visualized by confocal microscopy or analyzed by Flow cytometer.