Choosing a fluorophore for the FL3 channel on a FACSCalibur™

The Becton Dickinson FACSCalibur™ is a two laser, four color instrument. A variety of fluorophores can be used on all of the channels, as described in the table below. In general, the choice of the fluorophores for FL1, FL2, and FL4 do not present any complications (that's not to say that they don't require compensation!). In contrast, the choice of a fluorophore for the FL3 channel can present (avoidable) complications. This page is intended as a guide for choosing a fluorophore for the FL3 channel.

NOTE -- if you are using FL3 as an exclusion channel, it doesnt much matter which fluorophore you use.

Fluorophores for the FACSCalibur
Laser	Channel	Band Pass Filter	Common Fluorophore	Alternative Fluorophores
Blue (488 nm)	FL1	530/30	FITC	Alexa 488, GFP
	FL2	585/42	PE	-
	FL3	670LP	PerCP	PE-Cy5, PE-Cy5.5, PerCP-Cy5.5, PE-Alexa647, PE-Alexa680, PE-Alexa700, PE-Cy7, PE-Alexa750
Red (633 nm)	FL4	661/16	APC	Cy5, Alexa647

The source of the problem

Four factors must be considered in the choice of the FL3 fluorophore: (1) the optical bench of the Calibur, (2) the excitation and emission spectra of the various fluorophores, (3) the light sensitivity of the acceptor fluorophores in tandem dyes, and (4) factors which affect cross-beam compensation.

Optical bench

The optical bench is shown in the figure below (taken from the BD Introduction to Flow Cytometry: A Learning Guide). The key things to note are that the filter in front of the FL3 detector is a 670LP, while the filter in front of the FL4 detector is a 661/16 band pass.

Excitation and emission spectra

Here, the "problem" is that the acceptor dyes in many of the tandems are excited by the red laser (Cy5, Cy5.5, Alexa647, Alexa680, Alexa700); this means that PE (or PerCP) tandems incorporating these acceptors are excited by both the 488 and 633 laser on the FACSCalibur.

For PE-Cy5 and PE-Alexa647, excitation of the acceptor dye (Cy5 or Alexa647) is a problem, because these dyes have emission spectra that are very similar to APC, and their fluorescence is detected in the FL4 channel. As a result, use of these reagents requires extensive cross-beam compensation.

For the other tandems on the list (e.g. PE-Cy5.5, PE-Alexa700, PE-Cy7, PE-Alexa750), the acceptor fluorophore emits light at a longer wavelength than APC, and this light does not make it through the 661/16 bandpass filter in front of the FL4 detector. As a result, use of these reagents does not require extensive cross-beam compensation. This alone makes them superior (on the FACSCalibur) to reagents that do.

Light sensitivity of the tandem acceptor

All of the acceptor dyes used in PE (and PerCP) tandems are light sensitive. Of course, exposure to light may reduce the fluorescence intensity, diminishing the effectiveness of the stain. However, diminished fluorescence intensity is not the major drawback of the light sensitivity, since most of the dyes are very bright.

The major drawback of the light sensitivity of the tandems is that exposure to light changes the amount of fluorescence compensation required for the tandem reagents. This is a problem because compensation settings (or data files) are typically required at the beginning of an instrument session, so the compensation samples may be subjected to considerably less light-induced degradation of the tandem dye than are samples acquired later in an instrument session. Of course, this difference can be minimized by keeping all samples dark (in a covered ice-bucket), and attempting to equalize whatever light exposures is absolutely necessary.

The effect on compensation settings due to light induced degradation of the fluorophore is a factor for all tandem dyes, but especially so for those that require compensation versus FL4 (e.g. PE-Cy5, PE-Alexa647). For this reason, I recommend that you use alternative tandems.

Factors affecting cross-beam compensation

On all instruments with spatially separated lasers, a time delay is used so that data from the same cell detected by excitation by different lasers can be combined into a single "event". It is essential that the time delay be both accurate and stable over the course of an instrument session. On the FACSCalibur, the value of the time delay is automatically set when FACS Comp is run, and there is no way for the user to change it in CellQuest.

The time delay is sensitive to changes in sheath pressure. Unfortunately, on the FACSCalibur, the sheath pressure almost certainly changes as the sheath tank levels drop during an extended instrument session. As a result, the synchrony in data collected in the channels from the two different lasers is reduced. This will have a small effect on the signal intensity of the APC reagent. Unfortunately, it may have a large effect on the compensation of tandem dyes versus the FL4 channel. Again, this is a major factor for PE-Cy5 and PE-Alexa647, but probably a minor factor for the tandems that are not detected in the FL4 channel.

Finally, changes in sheath pressure during a run are probably much less of a factor on systems equipped with the FACS Flow fluidic cart. These systems pump sheath fluid into an intermediary "plenum", and the liquid levels (and therefore pressure) in the plenum remain constant.

Recommendations

So which fluorophore should you use for FL3 on the FACSCalibur?

First of all, unless you are using FL3 as an exclusion channel, avoid the use of PE-Cy5 and PE-Alexa647. If you are using FL3 as an exclusion channel—and if your reagents in FL3 give low backgrounds—then it doesn't much matter what you use.

Years ago—before the commercial availability of additional tandems—we switched to PerCP, which continues to perform well. However, it has the drawback that PerCP reagents tend to be very expensive. A few years ago, BD introduced the PerCP-Cy5.5 tandem, which they claim is brighter than PerCP. However, we normally use a very bright antibody in the FL3 channel (e.g. CD8), and we have never been limited by the signal intensity of PerCP. It has never made sense to me to make the switch to a reagent that is bound to introduce complications.

In the last couple of years, suppliers have begun to fill their catalogs with PE-Cy5.5, PE-Cy7, and now PE-Alexa700 and PE-Alexa750 tandems. In particular, keep an eye on Caltag, which was recently bought by Invitrogen—which also bought Molecular Probes—and which is touting PE-Alexa700 as the "superior FL3 fluorophore" (PDF) . This is not just marketing hype.

In summary, I recommend that you consider both PerCP and PE-Alexa700 (or PE-Cy5.5) fluorophores for the FL3 channel on the FACSCalibur. For a given specificity, compare both performance and price. In generally, I expect the PE-Alexa700 tandems to be cheaper, and to perform as well, if not better, than PerCP

These recommendations are summarized in the table below.

Summary of Dyes for FL3 Channel on the FACSCalibur
Dye	Excitation by Red Laser	Detection through 661/16 Filter on FL4	Comments
PerCP	No	Not relevant	Recommended (though expensive due to single supplier - BD)
PerCP-Cy5.5	Yes	Minimal	BD recommends this, and says it's brighter than PerCP. However, I don't get the point of using the PerCP tandem; in my mind, it doesn't add that much.
PE-Cy5	Yes	Yes	Once the standard FL3 reagent; now not recommended. (Known by many tradenames, including CyChrome, Tricolor, and PC5).
PE-Alexa647	Yes	Yes	No better than PE-Cy5. Not recommended.
PE-Cy5.5	Yes	Minimal	Recommended. May be superseded by PE-Alexa700.
PE-Alexa680	Yes	Mimimal	Not bad. But most suppliers seem to be gravitating towards PE-Alexa700.
PE-Alexa700	Yes	Minimal	Recommended.
PE-Cy7	Yes	No	Probably not at bright as PE-Cy5.5, PE-Alexa680, or PE-Alexa700. But it will work just fine.
PE-Alexa750	Yes	No	Probably not at bright as PE-Cy5.5, PE-Alexa680, or PE-Alexa700. But it will work just fine.

What about "FL3" reagents for the LSR-II and Aria?

On the LSR-II, there is more than one detector associated with the red laser. As a result, the red laser can excite the Cy5.5 or Alexa700 attached to PE tandems, and this light will be detected in the "Alexa700" channel associated with the red laser.

Based upon this factor alone, my bias is that when you are using 4 fluorophores off the blue laser, PerCP is a significantly better choice than PE-Cy5, PE-Cy5.5, or PE-Alexa700 (the combination I recommend is FITC, PE, PerCP, and PE-Cy7). When you are using 5 or 6 colors, you may be forced to use PE-Cy5.5 or PE-Alexa700 tandems, and to live with the compensation.