Ki67 fitc monoclonal antibody sola15

5,000 cells of the 5637 cell line were plated on 6-well plates. The following day, cells were serum starved for 24 hours to synchronize the population, after which media containing DMSO and 100 nM T0070907 were added. Cells were harvested after 5 days of drug treatment, fixed using ice-cold 70% ethanol, and stored at −20°C for 2 hours. Following centrifugation and removal of ethanol, cells were washed twice in FACS buffer (PBS containing 2% FBS and 1 mM EDTA). For dual Ki67/PI staining to determine non-proliferative (G₀ population): samples were resuspended in 200 μL FACS buffer and stained for 30 minutes at RT in the dark with Ki67-FITC monoclonal antibody (SolA15) (Life Technologies, cat. 11-5698-82) at 1:100. Cells were then washed twice with FACS buffer and resuspended in 500 μL PI staining solution (PBS containing 50 μg/mL PI, 100 μg/mL RNAse A and 2 mM MgCl₂). Cells were strained through 40 μm filters and incubated at RT in the dark for 20 minutes before analysis by flow cytometry. For analysis of the cell cycle phases, cells were stained with PI antibody only.

2 X 10⁵ cells were plated on 6-well plates and grown in full medium. The next day, the cells were washed twice with PBS and supplemented with either full medium, or Arg free medium, which was changed 48 hrs later. Cells were harvested the next day (72 hrs), fixed using ice-cold 70% ethanol, and stored at −20°C for 2 hrs. Following centrifugation and removal of ethanol, cells were washed twice in FACS buffer (PBS containing 2% FBS and 1 mM EDTA). For dual Ki67/PI staining to determine non-proliferative (G0 population): samples were resuspended in 200 μL FACS buffer and stained for 30 minutes at RT in the dark with Ki67-FITC monoclonal antibody (SolA15) (Life Technologies, cat. 11-5698-82) at 1:100. Cells were then washed twice with FACS buffer and resuspended in 500 μL PI staining solution (PBS containing 50 μg/mL PI, 100 μg/mL RNAse A and 2 mM MgCl2). Cells were strained through 40 μm filters and incubated at RT in the dark for 15 minutes before analysis by flow cytometry.