Z2 cell and particle counter

Dictyostelium Ax2 wild type and transformed cell lines were grown to exponential phase in axenic liquid media, complemented with the appropriate antibiotics. Cells were diluted to a density of 5x10⁴ cells per mL in fresh medium. Cell numbers were determined using a Beckman Coulter Z2 Cell and Particle Counter at 12–hour intervals over a period of 72 hours.

Cells (100 000/well) were seeded to the wells of a 6‐well plate. After overnight incubation, culture medium was replaced with fresh medium supplemented with designated reagents (vehicle control, JQ1, 3‐MA and etc). After 48 hours of treatment, cells were scraped and suspended, PBS was then added to make a final volume of 5 mL. The Beckman Coulter Z2 cell and particle counter (Indianapolis, IN, United States) was then used to determine the cell number.

For growth curve analysis, exponentially growing yeast cultures were inoculated into 10 ml fresh YPD media at density 5 × 10⁶ cells per ml. Cells were grown further in a shaker at 30 °C and samples were collected at indicated time-points during the growth. Culture density was measured with Z2 Cell and Particle Counter (Beckman Coulter). For spot test assays, 10-fold serial dilutions of cell suspensions were made and 5 µl of each dilution was spotted onto plates with synthetic complete (SC) selective medium. For plasmid shuffling, 1 mg/ml 5-fluoroorotic acid (5-FOA) and indicated concentrations of MMS in SC plates were used to test viability of cells. In experiments with Rpb9 anchor-away strains, 1 µg/ml rapamycin (Cayman Europe) in 0.1% DMSO as a final concentration was added to the cultures (0.1% DMSO was used for controls). Plates were incubated at least 2 days at 30 °C. For flow cytometry analysis of cell cycle, 0.5 ml of yeast culture was fixed in 10 ml of ice-cold 70% ethanol for at least 15 min and washed once with 50 mM citric acid. RNA was degraded with RNase A (10 μg/ml) in 50 mM citric acid overnight at 37 °C. DNA was stained with 10× SYBR Green I (Invitrogen) in 50 mM citric acid for 30 min. Cells were analysed with FACS Aria, cell cycle distribution was analysed with Cyflogic software.

Culture density was measured with Z2 Cell and Particle Counter (Beckman Coulter). For spot test assays, tenfold serial dilutions of cell suspensions were made and 5 µl of each dilution was spotted onto plates with synthetic complete (SC) selective medium. Indicated concentrations of methyl methanesulfonate (MMS) in SC plates were used to test the viability of cells. Cells were also treated with ionizing radiation (45–85 Gy). In experiments with Rpb9 anchor-away strains, 1 µg/ml rapamycin (Cayman Europe) in 0.1% DMSO as a final concentration was added to the cultures (0.1% DMSO was used for controls). Plates were incubated for 2 days at 30 °C, unless otherwise stated.

HeLa cells were plated in 6-well plates 16 hour prior to transfection at a concentration of 100,000 cells per well. Cells were transfected with control LNA, let-7 family power inhibitor LNA or miR-34a-5p LNA (Exiqon) at 15 nM per well in triplicates. Cells were reseeded at a concentration of 50,000 cells per well 24 hour post transfection and treated with 6.5 μM VLX600 or DMSO. Cells were allowed to expand for two and four days before assayed for absolute cell number counts using a Beckman Coulter Z2 Cell and Particle counter. Cells in a diameter range from 12 to 30 μm were counted.