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Flow cytometry and cell cycle analysis

EG Eda Gjika
SP Sonali Pal-Ghosh
MK Megan E. Kirschner
LL Li Lin
JS Jonathan H. Sherman
MS Mary Ann Stepp
MK Michael Keidar
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DNA staining was performed for U87MG cells treated for 180 s with plasma and TMZ concentrations of 10 and 50 μM. After 6 days in culture following the procedure described in “CAP and temozolomide treatment”, U87MG cells were harvested and centrifuged at 1200 rpm for 5 min. They were further washed 2× with phosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol and stored at 4 °C for 2–4 days. Cells were washed 2× with PBS and incubated in the dark in PBS containing 20 mg/ml RNase A (Thermo; EN0531) and 50 mg/mL propidium iodide (PI) (Thermo; P3566) at 37 °C for 30 min. Flow cytometry data collection was performed on a BD Celesta Cell Analyzer at the George Washington University’s Flow Cytometry Core Facility. Data analysis for DNA content was performed with FCS 6 Express software (De Novo; Glendale, CA) for quantifying G1, S and G2/M-phases of the cell cycle.

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