To analyze cell-cycle phases distribution, both floating and adherent DOX-untreated D283-OMO cells were collected by centrifugation, fixed in cold 70% ethanol, and then stained in a PBS solution containing propidium iodide (PI; 62.5 μg/mL; P4864, Sigma-Aldrich), and RNase A (1.125 mg/mL; R6148, Sigma-Aldrich). Cell aggregates were gated out on bi-parametric graph FL-3lin/ratio as described [30 (link)]. Cell samples were analyzed in a Coulter Epics XL cytofluorometer (Beckman Coulter, Brea, CA, USA) equipped with EXPO 32 ADC software. At least 10,000 cells per sample were acquired. The percentage of cells in the different phases of cell-cycle and in sub-G1compartment was calculated using Flowing Software 2.5.1.
Apoptosis induction was analyzed by flow cytometry determination of Annexin V-FITC staining (556420, BD Biosciences, Franklin Lakes, NJ, USA)/PI (P4864, Sigma-Aldrich), to label necrotic or late apoptotic/dead cells with damaged cell membranes. Cell samples were analyzed as already described for cell cycle analysis.
Apoptosis induction was analyzed by flow cytometry determination of Annexin V-FITC staining (556420, BD Biosciences, Franklin Lakes, NJ, USA)/PI (P4864, Sigma-Aldrich), to label necrotic or late apoptotic/dead cells with damaged cell membranes. Cell samples were analyzed as already described for cell cycle analysis.
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