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 flow 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

Note

• Alternatively, E. coli cells can be stained using a membrane dye, FM 4-64 as described in Evolutionary Stability of Fluorescent Protein Expression, Measurement by Flow Cytometry

Single samples

• When running individual samples on the Fortessa, use 5mL polystyrene round-bottom tubes (catalog number = BD Falcon 352058).
• Once samples have been stained and resuspended, dilute each sample 1:1000 in 1mL of saline/PBS.
• It's good practice to clean the instrument before running your samples, follow the detailed instructions that are posted on the Fortessa for cleaning. Briefly, run 10% Contrad (cleaning solution), bleach, and then DI water, each for one minute.
• Load your samples on the Fortessa:.
  • your entire population should appear in the FSC vs SSC plot, if not change the FSC/SSC gain settings until you see the whole population
  • run positive and negative controls first to ensure that the fluorescence gain values are set correctly
  • make sure that the event rate is in between 1000-2000 event/second
  • generally, acquiring ~10,000 total events is sufficient to get a good distribution of your population
• Once all of your samples have been analyzed, export your raw data as .fcs files, upload to UT FlowStation and use FlowJo for downstream analysis.
• Don't forget to clean the instrument again!

High Throughput, 96-well plates

• Connect the HTS (BD High Throughput Sampler) unit to the Fortessa
• Load HTS cleaning solution and water in an empty 96-well plate. Run the HTS cleaning protocol, it takes ~15 minutes.
• Put your plate in the HTS unit and run on High Throughput mode
  • Your samples need to be diluted sufficiently in order to obtain 10,000 events at a 1000-2000 events/second rate
  • Each plate takes ~22 minutes to analyze using the high throughput mode
  • Alternatively, plates can be run using the standard mode - make sure to initially run controls to properly calibrate instrument settings
  • To test for cross-contamination between wells, randomly include PBS blanks in between your samples
• Once your plates have run, clean the instrument again (this step takes ~15 minutes) and disconnect the HTS unit from the Fortessa.

-- Main.DanielDeatherage - 02 Jul 2012