Conjugates of these rhodamine derivatives have different absorption (550, 570, and 596 nm) and emission (570, 590, and 620 nm) maxima (Table 1 and Figure 2). Although TRITC has been commonly used with FITC for double labeling, better color separation is achieved by using RRX or Texas Red. However, it has been reported that use of Texas Red may lead to higher background staining (Wessendorf and Brelje, Histochemistry. 1992. 98, 81).

Table 1: Approximate* peak wavelengths of absorption and emission for different fluorophore-conjugated, affinity-purified antibodies.

*Only approximate values are given for purposes of comparing one fluorophore with another.
Actual values may vary depending on the spectrofluorometer used in each laboratory.

Excitation	Emission

Rhodamine Red-X is particularly useful for triple labeling with Cy2 (or FITC) and Cy5 using a confocal laser scanning microscope equipped with a krypton/argon laser, because fluorescence from RRX lies about midway between that of Cy2 and Cy5 and shows little overlap with either (see spectra below). The krypton-argon laser emits lines at 488 nm, 568 nm, and 647 nm, all of which are optimal for exciting Cy2 (or FITC), RRX, and Cy5, respectively.

Excitation (EX) and emission (EM) spectra of       Rhodamine Red-X conjugated antibody.	Emission spectra of cyanine- and Rhodamine       Red-X (RRX)- conjugated antibodies.

For double labeling with FITC in flow cytometry, phycoerythrin (PE) conjugates are recommended rather than rhodamine conjugates, since both FITC and PE can be excited by the 488 nm line of an argon laser. Flow cytometers typically do not have lasers which will excite any of these rhodamine dyes.