Facs aria fusion 2 cytometer

The Annexin V-FITC Apoptosis Detection Kit (Sigma) was used to identify the amount of exposed phosphatidylserine of S. vesiculosa M7^T cells at different times of growth to investigate apoptosis. Two 1ml aliquots of each sample were centrifuged at 10,000g for 10min at 20°C and washed twice in DPBS. Cells were resuspended and diluted to 10⁶ cells/ml in 1×Binding Buffer (BB). The Annexin V-FITC was added to each sample at a final concentration of 0.5μg/ml. Propidium iodide (PI) was also added to the samples at a final concentration of 2μg/ml, and they were incubated for 10min at room temperature in the dark. Stained cells were analyzed with the BD FACS Aria^™ Fusion II cytometer (BD Bioscience). Several negative controls were used to optimize the analyses: 1×BB, 1×BB with both the fluorochromes, and 1×BB with S. vesiculosa M7^T cells without fluorochromes. This experiment was carried out with each condition in duplicates in four independent experiments.

TheAPO-BrdU^™ TUNEL Assay (Invitrogen) was performed on the bacteria to analyze their cellular DNA damage at different times of growth. Two 1ml aliquots of each sample were diluted to 10⁷ cells/ml in DPBS in a final volume of 500μl. The bacterial suspension was added to 4.5ml of 2% (w/v) paraformaldehyde and incubated for 15min on ice. The cells were then centrifuged at 10,000×g, at 20°C and the supernatant was discarded. The pellet was resuspended in DPBS and washed twice. The cells resuspended in 100μl of DPBS were added to 900μl of 70% (v/v) ethanol and kept at −20°C for 12h. The alcohol suspension was centrifuged at 10,000g for 5min to remove the alcohol, and the pellet was resuspended in 50μl of the labeling solution. The cells were incubated at 37°C in the dark for 4h and shaken every 15min. Washing buffer (450μl) was then added and centrifuged at 10,000g for 5min. The pellet was resuspended in 100μl of the kit antibody solution. The cells were incubated at room temperature in the dark for 30min. A total of 500μl of the staining solution containing RNase A and PI was added to the resuspended cells and incubated at room temperature in the dark for 30min. Cell analysis was performed on the BD FACS Aria^™ Fusion II cytometer (BD Bioscience). This experiment was carried out with each condition in duplicates in two independent experiments.

MVs were isolated and stained with both SYBR^™ Gold and FM4-64 to separate MVs from those only stained with FM4-64 to identify the ones with internalized nucleic acids. A 70-μm nozzle was used on the BD FACS Aria^™ Fusion II cytometer (BD Bioscience) for separation. First, the controls (sterile and filtered DPBS, sterile and filtered DPBS with FM4-64, and sterile and filtered DPBS with FM4-64 and SYBR^™ Gold) and samples to be separated were analyzed to verify the percentage of events detected to have both fluorochrome labels and to identify the range of events/s. Then, two sterile 15ml collecting tubes (TPP^®, Merk) containing 100μl of sterile water were used to collect each separated sample. PBS FacsFlow^™ was used as the dilution buffer used during the sorting process (Fischer Scientific, United Kingdom). Once the sorting started, the injection flow was monitored and adjusted to keep the separation efficiency above 85%. The collected MVs were transferred to 8ml polycarbonate centrifuge tubes (Beckman Coulter) and centrifuged in the OPTIMA^™ L-90K ultracentrifuge (Beckman Coulter) with Ti/70 rotor at 100.000xg for 90min at 4°C. Supernatants were discarded, and pellets were resuspended in 20μl of sterile water and kept at 4°C until fixation for Cryo-EM.