Influx fluorescence activated cell sorter facs

All chemicals used for media and other experiments are from Sigma (St. Louis, MO) unless otherwise noted. Synechocystis 6803 cultures of WT, CB, CK, and PAL were initially inoculated into 20 ml of liquid TES-buffered BG11 medium [19 ] and grown for 7 days in a Sanyo Versatile Environmental Test Chamber at 30°C under 20 μmol photons m^-2 s^-1 white fluorescent light with constant shaking at 125 rpm. Due to the different growth rates among the strains, cultures were sub-cultured based on cell concentration. The concentration of Synechocystis 6803 cultures were determined by flow cytometry using a BD Influx Fluorescence Activated Cell Sorter (FACS, BD Biosciences, San Jose, CA). Upon harvesting, 100 ml of each sample was analyzed using the 488-nm laser excitation from a Sapphire LP laser (Coherent Inc., Santa Clara, CA) at 100 mW. Forward and side scatter were used to gate out cellular debris and values are recorded. Optimization and calibration of the FACS was performed before each analysis using 3 μm Ultra Rainbow Fluorescent Particles (Spherotech, Lake Forest, IL). Cultures were then grown for 5 days, and harvested by centrifugation for proteomics analysis. Growth medium was supplemented with antibiotics as follows: CB and CK, 10 μg/ml kanamycin; PAL, 10 μg/ml chloramphenicol and spectinomycin [20 (link)].

Cell populations were enriched by flow cytometry using the BD Influx Fluorescence Activated Cell Sorter (FACS, BD Biosciences, San Jose, CA, USA). Using the 488-nm to trigger cells, samples were analyzed using a 100-μm nozzle. Optimization and calibration of the FACS was performed before each analysis using 3-μm Ultra Rainbow Fluorescent Particles (Spherotech, Lake Forest, IL, USA). Forward and side scatter detectors were used to gate out cellular debris. The 561 nm laser was used to excite mCherry while measuring emission at 585 nm with a 29 nm bandpass filter. Cell populations were selected for enrichment in mCherry signal. Median calculations from 10,000 cells were done using Flow Jo software (Tree Star, Ashland, OR, USA).