2008-04-02
- Diluted 50 μl of overnight (20 h) culture into 5 ml of pre-warmed (37°C) fresh media (1:100)
- Grew for 4 h at 37°C with shaking at 225 RPM
- Note that I used a higher RPM than mentioned in the protocol
- Observed turbidity visually after 4 h
- Pelleted 1 ml of each culture in a 1.5 ml microcentrifuge tube at 3000 RCF for 5 min at RT
- Pellet sizes were all similar except for the three I20260 cultures, which all appeared smaller
- Stored remaining cultures at 4°C for future OD600 readings (if necessary)
- Removed the supernatant
- Resuspended the cell pellets in 1 ml of 1X PBS
- Added 500 μl of resuspended cells through a cell strainer lid into a 5 ml polystyrene tube
- Placed cells on ice
- Analyzed cells by flow cytometry using a BD FACSCalibur (15 mW 488 nm, air-cooled argon-ion laser) within 30 min
- Captured 250,000 events per sample (this seems to be far more than necessary)
- Observed fluorescence in the FL1 channel (530/30 BP emission filter)
- Used the following instrument settings (threshold was not optimized for samples):
Detectors/Amps:
Param Detector Voltage AmpGain Mode
P1 FSC E03 1.00 Lin
P2 SSC 551 1.00 Lin
P3 FL1 690 1.00 Log
Threshold:
Primary Parameter: FSC
Value: 52
Secondary Parameter: None
- Raw FCS data files are here
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