Alexa fluor 647 conjugated anti mouse antibody

Flow cytometry was conducted on a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data analyzed with flowjo software (version 10.6.1; BD Biosciences, Franklin Lakes, NK, USA; RRID: SCR_008520). For cell number and viability counts, cells were resuspended in ice‐cold PBS in the presence of 1 µg·mL⁻¹ Propidium Iodide (P.I.). Cell viability is given as percentage of live cells on total cells counted, while the proliferation index is defined as the ratio of viable cells in the sample to viable cells in the corresponding IACS‐010759‐untreated control.
For evaluation of apoptotic cell death, cells were stained with APC‐conjugated Annexin V (Thermo Fisher Scientific) and P.I. as previously described [11 (link)]. The cytoplasmic release of cytochrome c was detected as described [38 (link)] with few modifications: Briefly, cells were permeabilized with 0.002% digitonin for 20” under vortexing before fixation in 4% PFA and permeabilization with 0.1% Triton X‐100, for subsequent immunostaining with anti‐cytochrome c (clone 6H2.B4, BD Biosciences) overnight at 4 °C. Secondary staining was performed with an Alexa Fluor 647‐conjugated anti‐mouse antibody (Jackson ImmunoResearch).

Immunocytochemistry analysis was performed according to a previous report^{21 (link)}. The cells were seeded onto slides. At 70% confluence, the cells were fixed in 4% paraformaldehyde in PBS buffer for 15 min. Following washes with PBS, the cells were permeabilized in freshly prepared 0.1% Triton X-100 for 10 min and blocked in 1% bovine serum albumin (BSA) in PBS buffer for 1 h at room temperature. The cells were incubated with primary antibodies directed against either runx2 (1:200, ab76956, Abcam), the α-subunit of BK (1:100, ab99046, Abcam, USA), integrin beta1 (1:100, 1798-1, Epitomics, USA), Flag (1:200, Rabbit Monoclonal Anti-Flag antibody, F2555, Sigma) or His (1:200, SAB1306084, Sigma) at 4 °C overnight. The secondary Alexa Fluor 647-conjugated anti-mouse antibody (Jackson Immuno Research, USA) was used at a 1:200 dilution for 2 h at room temperature protected from light. Following washes with PBS, the cells were stained with DAPI for 5 min. The slides were mounted with SlowFade™ Gold Antifade Mountant (S36936, Invitrogen), and images were captured using a Leica TCS SP5 confocal microscope (Leica, Germany) at room temperature with ×10 and ×40 objective lenses.