(Difference between revisions)
Revision as of 09:31, 5 May 2013
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.
- 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!!!).
- At least 100,000 events should be collected and stored for each sample during analysis.