Anti ki67 monoclonal antibody clone mib 1

Proliferation rate was defined as the percentage of Ki67+ cells out of total nucleated cells (Ki67%). Briefly, an intracytoplasmic labelling with a FITC-conjugated anti-Ki67 monoclonal antibody (clone MIB-1, DAKO) was performed; a commercial permeabilization (Poggi et al., 2015) . A control tube was prepared in the same way except for the addition of FITC-conjugated isotypic immunoglobulin instead of the anti-Ki67 antibody. Apoptotic rate was defined as the percentage of cells staining positive for Annexin V, but negative for Propidium Iodide (AnnV%). A commercial kit was used to this aim (AnnexinV-FITC, Apoptosis detection kit, eBioscience), according to the manufacturer's instructions. Briefly, cells were suspended in 200 μl of Binding Buffer with a concentration of 2 × 10 5 cells/tube and incubated for 10 min at room temperature with 5 μl of anti-AnnexinV-FITC antibody. Thereafter, 10 μl of propidium iodide (PI) were added and incubated for 5 min before acquisition at the cytometer. A negative control with unstained cells was also acquired for each sample. Ki67% and AnnV% were finally coupled to calculate PAR (Ki67% / AnnV%) and TI (Ki67% + AnnV%).