By far the most commonly used fluorophores for MHC tetramers are the algae-derived phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC).  Conjugates of these fluorescent proteins with streptavidin are widely available from a number of commercial suppliers, and these fluorophores provide some of the best signal-to-noise ratios on most flow cytometers.  The NIH Tetramer Facility exclusively uses streptavidin-PE and streptavidin-APC from Molecular Probes (now Invitrogen), and over the years these reagents have proven to be extremely robust.  Other labs making their own tetramers have used streptavidin reagents from other suppliers, also with great success.

With the development of multicolor flow cytometers such as the LSR-II from Becton Dickinson and the development of protocols such as flow FISH that require harsh denaturing conditions, there is an increasing demand for tetramers prepared with small molecule fluorophores.  Until recently, the NIH Tetramer Facility has had only intermittent success in preparing MHC tetramers with dyes such as fluorescein, Cy5, Alexa647 or Pacific Blue.  However, we have recently taken advantage of mutant recombinant streptavidins engineered by Takeshi Sano and colleagues to contain a single cysteine residue at the carboxy-terminus of each subunit of the streptavidin homotetramer to effectively produce MHC tetramers incorporating small molecule fluorophores.

We are now offering tetramers with the following fluorophores:  Pacific Blue, fluorescein, Alexa 488, Alexa 647 and Alexa 680.  All are capable of providing baseline separation of tetramer-positive cells from tetramer-negative cells, though Pacific Blue tends to be the least bright while Alexa647 tends to be the brightest.  Please note, the emission spectra of fluorescein and Alexa 488 are very similar, and these reagents cannot be used together.  Similarly, the emission spectra of Alexa 647 and APC are very similar, and these two fluorophores should not be used together.