Cells were treated with isotype control or specific antibodies for 30 min at 4 °C. Cell Signaling (Beverly, Massachusetts, USA) provided the human SOX2 antibody, while the anti-OCT4-PE antibody came from Miltenyi Biotec [43 (link)]. CellQuest software was used to evaluate cells employing a FACS Calibur cytofluorimeter (BD Biosciences, Franklin Lakes, NJ, USA; www.bdbiosciences.com ). Each sample covered at least 104 events that were recorded. Cells were permeabilized when required using the Cytofix/Cytoperm kit from BD Biosciences [28 (link)].
Cells were permeabilized with cold 70% ethanol in PBS for cell cycle analysis and stored at −20 °C until next day. Cells were then resuspended in PBS supplemented with 50 μg/ml propidium iodide (PI) and 100 mg/ml RNase, and were kept for 30 min at 30 °C. CellQuest software was used to analyze the data obtained through the FACS Calibur [28 (link)].
Dead cells were identified and distinguished using the Annexin V-FITC Kit from Miltenyi Biotec. Cell staining was performed in a dark environment for 15 min at room temperature by adding 10 μl of Annexin V-FITC to 106 cells, following a serum starvation of 18 h. Prior to analysis employing CellQuest software and the FACS Calibur, 5 μl of PI solution was added [28 (link)].
Cells were permeabilized with cold 70% ethanol in PBS for cell cycle analysis and stored at −20 °C until next day. Cells were then resuspended in PBS supplemented with 50 μg/ml propidium iodide (PI) and 100 mg/ml RNase, and were kept for 30 min at 30 °C. CellQuest software was used to analyze the data obtained through the FACS Calibur [28 (link)].
Dead cells were identified and distinguished using the Annexin V-FITC Kit from Miltenyi Biotec. Cell staining was performed in a dark environment for 15 min at room temperature by adding 10 μl of Annexin V-FITC to 106 cells, following a serum starvation of 18 h. Prior to analysis employing CellQuest software and the FACS Calibur, 5 μl of PI solution was added [28 (link)].
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