Alexa fluor 594 conjugated antibody

After reaching over 80% confluence, chondrocytes were shifted to serum-free culture medium for 24 h, and incubated in complete culture medium with 10ng/mL IL-1β for different time periods. Then, PBS was used to wash the cells and cold methanol was added for 20-min to fix the cells. Fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and washed with PBS. Then, a primary antibody directed against LC3 was utilized to treat cells overnight at 4°C in PBS with 1% BSA, then a secondary Alexa Fluor® 594-conjugated antibody (Cell Signaling Technology) was added for 1 hour at room temperature. Cell nuclei were stained with DAPI (Thermo Fisher Scientific) for 5 minutes, then coverslips were mounted on glass slides, and cells were observed with a Leica fluorescence microscope.

Apoptosis and proliferation were assessed by immunofluorescence visualization of cleaved caspase-3 (CL-caspase3) and Ki-67-positive cells, respectively, as previously described [72 ]. H157 and AGS cells were transfected in 6-well plates with different combinations of plasmids using Lipofectamine 2000 (Invitrogen) and collected 24 hours afterwards to be distributed in Lab-Tek Chamber slides system, 2×10⁵ cells per chamber. After 72 hours of transfection, cells were fixed with 4% para formaldehyde for 20 minutes, washed with PBS, blocked with blocking solution (5% fetal bovine serum, 0.3% Triton X-100 in PBS) for 1 hour, incubated with anti-CL caspase3 primary antibody (1:400 dilution; Cell Signaling Technology, QLD, Australia) or anti-Ki67 (1:400 dilution; Cell Signaling Technology, QLD, Australia) in blocking solution overnight and further incubated with an anti-rabbit or anti-mouse (for CL caspase3 or Ki67, respectively) secondary antibody Alexa Fluor 594-conjugated antibody (1:1000 dilution; Cell Signaling Technology, QLD, Australia) and Hoechst 33258 nucleic staining (1:10000 dilution). The percentage of positive cells was determined by counting red fluorescent cells versus total cells using a fluorescent microscope (Olympus IX71).