Accuri c6 flow cytometer instrument

Apoptosis of lymphocytes was determined in peripheral blood (PB) by Annexin V Apoptosis Detection kit (BD Biosciences, United States) using the BD Accuri™ C6 Flow Cytometer Instrument. The PB was collected from patients and healthy controls in tubes containing EDTA. 50 μL of EDTA‑treated PB were incubated for 30 min at 4 °C in the dark with Annexin V and propodium iodide. The red blood cells were lysed using BD FACS Lysing Solution (Becton Dickinson, USA). The stained cells were then washed and resuspended in buffer solution. Approximately 30,000 stained cells in each sample were analyzed using the BD Accuri™ C6 Flow Cytometer Instrument (BD Biosciences). The lymphocytes were gated by setting the appropriate forward scatter/side scatter axes. Data were acquired, and data analysis was performed by the BD Accuri™ C6 software program.

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.