Safranin o

Joint tissue sections were deparaffinized in xylene for 15 min and hydrated in an ethanol series. For H&E staining, the sections were incubated with Harris hematoxylin (Sigma-Aldrich) for 10 min, washed in tap water, and dipped sequentially in 1% HCl, 0.2% NH₄OH, and eosin (Sigma-Aldrich) for 90 sec and washed. For safranin O staining, the sections were incubated in Weigert's iron hematoxylin (Sigma-Aldrich) for 10 min, washed in tap water for 10 min, incubated in Fast green (Sigma-Aldrich) for 5 min, dipped in 1% acetic acid 2 to 3 times, incubated in safranin O (Sigma-Aldrich) for 5 min and washed. For toluidine blue staining, the sections were incubated in toluidine blue for 3 min. Each stained section was subsequently washed in tap water, dehydrated in an ethanol series, dipped in xylene, and mounted. Inflammation and joint destruction scores were measured by three independent investigators. Inflammation scores were measured by adding scores, graded as 0-3, were based on the layer status of synovial membranes and scores, graded as 0-3, were based on the infiltration of lymphocytes. Joint destruction scores were measured by adding scores, graded as 0-3, were based on cartilage erosion, and scores, graded as 0-3, were based on pannus invasion into the cartilage. The detailed scoring method was described in reference study (19 (link)).

The Ca²⁺-induced modifications of mitochondrial transmembrane potential (ΔΨ) were measured by the evolution of fluorescence of Safranin O (Sigma-Aldrich) at 495 nm (excitation)/586 nm (emission) in the presence of energized mitochondria. The mitochondria were preincubated for 2 min in the chamber of the Hitachi F-3010 fluorometer at 37°C with a solution containing 320 mM mannitol, 10 mM Tris HCl (pH 7.4), 8 mM Tris-phosphate (H₃PO₄ neutralized with Tris base up to pH 7.4), 4 mM MgCl₂, 1 mg/ml BSA fatty acid-free (Sigma-Aldrich), 1 µM rotenone (Sigma-Aldrich), and 5 µM Safranin O (Sigma-Aldrich), in the absence or presence of 1 mM ADP. After mitochondrial energization with 10 mM succinate and rapid decrease in fluorescence intensity, successive pulses of 10 µM CaCl₂ allowed measurements of total Ca²⁺ load ([Ca²⁺]_t) required for complete depolarization (completed by adding 1 µM FCCP) and of the time necessary for 50% recovery of the baseline (t_1/2).

To mimic the OA environment in in vitro cell culture conditions, TC28a2 cells were seeded in the center of the individual wells of 24-well plates at 1×10⁵ cells per well in a 10-µL volume. The cells were then allowed to adhere for 2 h in a cell culture incubator. Next, DMEM-HG supplemented with 1% insulin transferrin selenium-A (ITS-A; Invitrogen, Carlsbad, CA, USA) was added gently. IL-1β (R&D Systems, Minneapolis, MN, USA) was chosen to induce and maintain inflammatory conditions, and the concentration of IL-1β was 10 ng/mL, based on our previous results.15 (link) The cells were maintained for 5 d in the absence or presence of IL-1β and stained with 1% safranin O (Sigma) solution to visualize proteoglycan content produced by the chondrocytes. Crystal violet (CV; Merck, Darmstadt, Germany) staining was performed to compare the density of the cells stained by safranin O. The CV staining method has been described previously.16 (link) For quantitative analysis, absorbance was detected at 490 nm after destaining with 50% acetic acid solution for 20 min. Each safranin O value was normalized to absorbance from CV staining.