We walked you through the basics of achieving your optimal panel design in the “Know your Flow” series, but you probably have more questions as you refine your panel or stumble upon some of the technical nuances of staining cells.
Check out these tips for a successful flow cytometry run.
1. How Many Cells?
Flow cytometry experiments “flow” best when you use a single cell suspension, and the ideal cellular density usually ranges from 1 X 105 to 1 X 106 cells /ml in 100 µl. How do you maintain your cells in a happy single-cell suspension state? You may need to strain cells during sample preparation. You will also typically use a solution of phosphate-buffered saline containing 1% bovine serum albumin or human serum throughout your staining protocol in order maintain cell viability and prevent cell clumping.
2. Fresh vs. Frozen Cells.
Many flow cytometry assays are done with cells isolated from fresh whole blood or tissue. In ideal circumstances, you can process, stain and run your your samples on a flow cytometer soon after collection. What if you are shipping your samples back to the lab from distant locations or you are collecting samples over several weeks or months and want to run them together? You may need to consider cryopreservation (freezing your cells to preserve viability) and will need to optimize your flow cytometry panel for cells that have been previously frozen and thawed. Take the time to work out ideal cryopreservation protocols, and find out if freezing will destroy your cells of interest.
3. Dead or Alive?
You’ll want to include a viability dye that discriminates between dead and live cells so you will only analyze live cells. Adding a viability dye may alter your staining panel because live/dead dyes use one of the channels on the cytometer that may have been reserved for a fluorescent antibody.
4. To Fix or Not Fix?
If you are staining for biomarkers on the cell surface, then you may be able to fix your cells with a cellular fixative like paraformaldehyde and run samples at a later time. If you are staining intracellular biomarkers, you need to perform a fixation/permeabilization step after you stain your surface markers so fluorescent antibodies can stain their intracellular targets. In either case, fixation alters cells and may change the way fluorescently conjugated antibodies stain their targets. Keep this in mind as you optimize your panel and set the appropriate gating parameters on the flow cytometer.