FL1 = Intracellular cytokine staining with eg. anti-IL2-FITC (home-made)
FL2 = "Naive" T-cells staining with anti-CD45RB-PE (Sigma)
FL3 = "Memory/activated T-cell staining with anti-PgP-1(CD44)-Quantum Red (a PE-Cy5 tandem dye from Sigma)
FL4 = "Helper" T-cell subset with anti-CD4-biotin followed by Streptavidin-Allophycocyanin (SA-APC from Pharmingen)
FL1 = "MLS-reactive" T cell receptor subset staining with anti-Vbeta6-FITC (home-made)
FL2 = "Helper" T-cells staining with anti-CD4-PE (Sigma)
FL3 = "Cytotoxic" T-cells staining with anti-CD8-Quantum Red (Sigma)
FL4 = Cytokine receptor (anti-CD25 or anti-IL4R) biotin conjugates (home-made) followed by SA-APC
Although the Cy5 in Quantum Red and also the APC are both excited by the red diode laser, the two signals are distinguished in time. In practice this means that as long as the time delay calibration is first correctly set up (we use CD4-biotin plus SA-APC labelled and fixed thymocytes for this - which is then automatically performed by the computer with an option from the Cytometer menu of CellQuest), then compensation of FL3-4% and FL4-3% is performed using single stained samples in an identical fashion to 3 colour compensation for FL1,2 and 3 (except you perform the adjustments from CellQuest as the FACSort has no front panel).
Here are some example instrument settings (for guidance only):
Detector Voltage AmpGain Mode FSC E00 3.28 Lin SSC 288 1.00 Log FL1 700 1.00 Log FL2 550 1.00 Log FL3 600 1.00 Log FL4 785 1.00 Log Threshold (FSC) = 144 Compensation: FL1 - 1.5% FL2 FL2 - 16.0% FL1 FL2 - 0.7% FL3 FL3 - 9.8% FL2 FL3 - 30.8% FL4 FL4 - 53.9% FL3
Note in this example that FL4 is set high to allow detection of weak FL4 fluorescence (eg IL4-R) so this is why FL3/FL4 compensation values are also high.
If you want to analyse lymphocytes and start with my standard intrument settings then aquiring 4 colour data should be as painless as single colour. (Mind you, sorting out what all the data means can be more tricky)!
Click here to see an example four colour cytokine staining of spleen cells (gated on Vbeta6-biotin/SAv-APC positive or negative and CD4-PE positive, and showing CD44-QR vs. anti-IFN-gamma-FITC).
NB. I have heard from more than one source that attempts to use this 4 colour system on machines other than upgraded FACSorts or FACSCaliburs (eg. FACSVantage) have been much more problematic.
More info soon but in the meantime please see Steve Cobbold for further details.