Fitc anti ki67 antibody

At 24, 48, 72, or 96 hrs after treatment with ribavirin (50 μM), cells were collected, washed in PBS and prepared for flow cytometry. For cell death, we used reagents from the APC-AnnexinV/Dead Cell Apoptosis Kit (Invitrogen). For cell cycle analysis, cells were labeled with FITC-anti-Ki67 antibody (Abcam, Cambridge, MA, USA) and run on a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA). Subsequent analyses were performed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Forty-eight hours after treatment with metformin (10mM), temozolomide (100μM) and/or ionizing radiations (5Gy), cells were collected, washed in PBS and prepared for flow cytometry. For cell death, we used reagents and protocols from the FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen Life Technologies). Briefly, we incubated cells for 15min at RT in 100μL of Annexin-binding buffer 5X (10mM HEPES pH7.4, 140mM NaCl, 2.5mM CaCl₂), containing 5μL of Annexin V-FITC antibody and 1μL of Propidium Iodide (PI) solution at 100μg/mL. 400μL of Annexin-binding buffer 5X were then added after washes with PBS-BSA 1%. For cell cycle analysis, cells were fixed with cold ethanol 100% and then permeabilized with Triton X-100 at 0.25%. Cells were then labeled with FITC-anti-Ki67 antibody (Abcam) during 45min at RT and treated with RNAse A at 1μg/mL before labeling with PI during 2hrs at RT. For autophagy analysis, cells were labeled with Acridine Orange (1:10000 dilution, Fluka) for 20min at RT and washed with PBS. Labeled cells were preserved on ice and run on a flow cytometer (FACS Calibur, Becton-Dickinson).