FACS Analysis of Cell Cycle

FACS experiments were performed as described previously50 (link). Cells in exponential growth phase (OD_660nm=0.3–0.6) or in stationary phase (diluted to obtain an OD_660nm=0.3–0.6), cultivated in M2G, were fixed in ice-cold 70% ethanol solution. Fixed cells were re-suspended in FACS staining buffer, pH 7.2 (10 mM Tris-HCl, 1 mM EDTA, 50 mM NaCitrate, 0.01% Triton X-100) and then treated with RNase A (Roche) at 0.1 mg ml⁻¹ for 30 min at room temperature. Cells were stained in FACS staining buffer containing 0.5 μM of SYTOX Green nucleic acid stain solution (Invitrogen) and then analysed using a BD Accuri C6 flow cytometer instrument (BD Biosciences). Flow cytometry data were acquired and analysed using the CFlow Plus V1.0.264.15 software (Accuri Cytometers Inc.). 20,000 cells were analysed from each biological sample. The forward scattering (FSC-A) and Green fluorescence (FL1-A) parameters were used to estimate cell sizes and cell chromosome contents, respectively. Experimental values represent the averages of three independent experiments. Relative chromosome number was directly estimated from the FL1-A value of NA1000 cells treated with 20 μg ml⁻¹ Rifampicin for 3 h at 30 °C, done in ref. 50 (link). Rifampicin treatment of cells blocks the initiation of chromosomal replication, but allows ongoing rounds of replication to finish.