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Protocol
Analysis
Some points to consider before/during collection and analysis:
- Acquire data in log mode to accommodate shifts in fluorescence (some epitopes show 30 fold or more increases in fluorescence upon phosphorylation).
- Acquire at least 10,000 events to obtain smooth histograms.
- A simple way to correlate staining intensities from day to day is to set an unstained, or isotype stained, sample to the lowest quadrant of the FACS plot. Good staining typically is close to unstained controls in unstimulated samples.
- Shifts between stimulated and unstimulated samples can vary greatly depending on the epitope of interest. Shifts from 2-fold to 50-fold have been observed. As more antibodies are validated for flow cytometry larger shifts will most likely be obtained.
- Be cautious when combining multiple colors as each color can shift in fluorescence. This is critical when compensating between, for example, FITC (Alexa 488) and PE, since the amount of leakage can vary depending on how much phosphorylation is present. Careful compensation must be performed prior to doing multi-color experiments of any kind (or performed with software such as FlowJo, www.treestar.com).
