Magni:Protocols/flowcy

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Preparation of samples for flow cytometry

• Grow cells under the desired conditions.
• 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

NOTES:

• 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!!!).