Notebook:Jason R. Kelly/2008/02/13

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FACS protocol V0.1

Materials

  • 5 strains from Jason Kelly
  • TOP10 background strain (Jason will provide so we're all actually using the same one).
  • 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS)
  • 5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235

Live Cell Procedure

  • When your 5 mL culture reaches OD600 = 0.100, pellet the cells by centrifuging at 3000xg at room temperature for 5 minutes.
  • Remove the supernatant.
  • Resuspend the cell pellet in 5 mL PBS.
  • Re-pellet the cells by centrifuging at 3000xg at room temp for 5 min.
  • Repeat wash with 5 mL PBS.
  • Re-suspend washed cells in 0.5 mL PBS. The cell concentration should be approximately 5x108 cells/mL.
  • Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
  • Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
  • Put cells on ice.
  • Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.