User:Dileep D. Monie/Notebook/2008-4-2

• 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 cells, which 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 withing 30 min
  • Captured 250,000 events per sample (this seems to be far more than necessary)
  • Observed fluorescence in the FL1 channel
  • 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