Atto 594 secondary antibody

To visualize proliferation marker, immunostaining with Ki-67 antibody (ab15580, Abcam, Cambridge, UK) was conducted. Cells were seeded on slides in 6-well plates. After 24 h incubation with the appropriate compounds, the medium was removed, and the cells were fixed with 4% PFA as described above. Then, samples were washed with PBS and were permeabilizated with 0.05% Triton X-100 in PBS for 15 min at room temperature in the dark. Treated and untreated cells were incubated overnight with the Ki-67 antibody diluted in 10% Goat Normal Serum (#31872, Invitrogen) in PBS at 4 °C (1:1000). Cells were washed with PBS 3 times and incubated with atto-594 secondary antibody (77671, Sigma-Aldrich/Merck) diluted in PBS for 1 h in the dark in the room temperature (1:1000). The cell nucleus was stained with DAPI (Faramount Aq Mounting Medium, Dako, Santa Clara, CA, USA). Cells were observed with a confocal microscope, and the obtained data were analyzed using Image J Software.

To visualize the proliferation marker Ki-67, immunostaining with Ki-67 antibody (Abcam, ab15580, Cambridge, UK) was performed. HuASCs were treated with the appropriate compounds for 24 h; then, the medium was removed and cells were fixed with 4% PFA as described above. Samples were washed with PBS twice and permeabilized with 0.05% Triton X-100 in PBS for 15 min at room temperature in the dark. Treated and non-treated cells were incubated overnight with the Ki-67 antibody diluted in 10% Goat Normal Serum (Invitrogen, #31872, Warsaw, Poland) in PBS at 4 °C (1:1000). Cells were washed with PBS three times and incubated with atto-594 secondary antibody (Sigma-Aldrich, 77671) diluted in PBS for 1 h in the dark at RT (1:1000). Cell nuclei were stained with DAPI (Faramount Aq Mounting Medium, Dako). Cells were visualized with a confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss, Germany), and the obtained data were analyzed using Image J Software (Bethesda, MD, USA).

Untreated and treated cells with LPS or/and mitochondria (MT1/MT2) were fixed with 4% PFA as described above. Cells were blocked with 5% BSA and 22 mg of glycine for 1 h at 4 °C and incubated overnight with the Ki-67 antibody (1:100, Abcam). Cells were washed with PBS 3 times following incubation with atto-594 secondary antibody (1:1000, Sigma). The cell nucleus was stained with DAPI. Cells were observed with a confocal microscope and the obtained data was analysed using Image J Software.