At the required time points, dilute cells 5-fold in filter-sterilized PBS 1X supplemented with gentamycin (final concentration of 1 mg/ml) in order to stop translation.
incubate the diluted cells under the same conditions as before for about 1 hour and then start analysis.
At least 100,000 events should be collected and stored for each sample during analysis.
After analysis, data can be gated and analyzed by the FloMax (Partec) software (only available in the same computer of the machine).
Otherwise, acquired data files can be imported in MATLAB via the fca_readfcs script (Laszlo Balkay). The acquired log-binned values of forward scatter (FSC), side scatter (SSC) and fluorescence (FL1) are integers in the range 0-4095. Log-binned FSC, SSC and FL1 values can be linearized, gated and background-subtracted as required
The available Partec PAS II flow cytometer is equipped with an argon ion laser using the 488 nm blue line for excitation and a 515-545 nm band-pass filter is used for emission. For this reason, only GFP can be detected.
At least 1 million of cells should be present in the final sample for analysis and the sample volume should be at least 1.5 ml. To do this, the 5-fold dilution can be adjusted as required. Cell count can be estimated by measuring the OD600 of culture and by using an OD600-CFU calibration curve (this is strain-dependent!!!).