Fortessa Flow Cytometry

• The Fortessa is a shared resource through the microscopy core. If you want to use it you need to get access to it you should fllow the directions provided by the microscopy core by talking to Richard Salinas.
• Other lab members may be able to help you initially set things up.
• Additional instruments with different capabilities are available.

General guidelines for distinguishing 2 different cell populations

• SYTO 17 dye is a red flourescent nucleic acid dye which stains all cells relatively equally.
• Attempts to optimize non-dye protocols were limited due to lack of knowledge of instrument settings (specifically ability to modulate threshold values of forward scatter). Can be revisited if would prefer not to use the dye.
• A final concentration of 135nM SYTO dye has been determined to be sufficient for REL606.
  • Concentrations above ~2.5 uM SYTO caused odd behavior (GFP positive cells read as GFP negative)
  • Optimization of SYTO may be worth while for different cell types or concentration of cells.
• Cell concentration determines the speed at which the instrument will read the samples. Too high a cell concentration and the instrument seems to have difficulty in distinguishing individual events (sloppier/more noise).
• ~2x10^4 cell/ul seems sufficient concentration to allow adequate range of adjustment for single samples on the instrument.
  • represents a 1:10 dilution of DM100

Single samples

• 5ml polystyrene round-bottom falcon tubes 352058 should be used for small (~12 samples), and 96 well plates should be used for larger number of samples. Plates run signficantly faster, but take slightly longer to clean the instrument after use, and provide less control on a per sample basis. Therefore plates work best when you know what you are expecting (GFP intensity

-- DanielDeatherage - 02 Jul 2012