Fc500 flow cytometry analyzer

For growth curve assay, primary astrocytes were seeded into 12-well culture plates at 200,000 cells/well in 0.5 ml of DMEM with pyruvate (10% FBS and 1% of streptomycin (10,000 µg/ml)-penicillin (10,000 units/ml)). Drugs were added to each well to obtain the desired concentration in a final volume of 1 ml per well. Day of seeding was considered day 0. Plates were incubated in a humidified incubator at 37 °C and 5% CO₂. Cells were harvested on each indicated day using 0.25% trypsin-EDTA (Gibco) and counted using an inverted phase contrast Zeiss microscope. For cell cycle analysis, Cells were plated at a density of 200,000 cells/well and attached overnight. The following day, the cells were deprived of FBS overnight to standardize the cell cycle followed by fresh DMEM containing 10% FBS and the indicated concentration of drug. At the indicated times, cells were harvested using 0.5% trypsin (Invitrogen) and washed with Wash Buffer (0.1% FBS in PBS) twice. Cells were fixed in ice cold 70% ethanol for 45 minutes at 4 °C. Ethanol was removed by washing twice with PBS, the cells were then incubated with propidium iodide (PI) (40 µg/ml) and RNAse (10 µg/ml) for 30 min at 37 °C. Samples were analyzed using Beckman Coulter FC500 Flow Cytometry Analyzer.

Astrocytes were seeded at a density of 40,000 cells/well in 12-well plates or 25,000 cells per well in 24-well plates with different culture medium. At the indicated days after seeding, cells were harvested using trypsin-EDTA and counted using a hemocytometer. Four wells were assigned to each group and cell counting was conducted by a researcher who was blinded to the group assignment using an inverted phase contrast Zeiss Invertoskop microscope.
For cell cycle analysis astrocytes were seeded at a density of 50,000 cells/well in 12-well plates in various culture media. On day 7 after culture, cells were harvested using trypsin-EDTA and washed with buffer (PBS) twice to remove trypsin. Cells were fixed in ice-cold 70% ethanol for 24 hours at 4°C. Then, cells were incubated with propidium iodide (PI) (40 μg/ml) and RNase (10 μg/ml) for 30 minutes at 37°C. The stained cells were analyzed using a Beckman Coulter FC500 Flow Cytometry Analyzer for quantification of cell cycle distribution (G1, S or G2/M).