The TCF offers tetramers labeled with fluorophores listed in the table below:
| Fluorophore | Absmax nm | Em nm | Lasers | Comment |
|---|---|---|---|---|
| PE | 496, 546, 565 | 578 | 488, 532, 568 | Brightest |
| APC | 650 | 660 | Red | Bright |
| BV421 | 405 | 421 | Violet | Bright replacement for PacBlue |
| Fluorescein | 494 | 518 | 488 | |
| Alexa 488 | 495 | 519 | 488 | We generally recommend this over fluorescein. |
| Alexa 568 | 578 | 603 | 532, 568 | Requires green or yellow-green laser |
| Alexa 647 | 650 | 668 | Red | We generally recommend starting with APC instead of Alexa 647, unless you need a small molecule fluorophore. |
| Alexa 680 | 679 | 702 | Red |
Streptavidin-PE and -APC are currently obtained from Agilent. Streptavidin-Brilliant Violet 421 (BV421) is obtained from BD Biosciences. Streptavidins labeled with the small molecule fluorophores (fluorescein, all of the Alexa dyes) are prepared in-house from a recombinant mutant streptavidin containing a C-terminal cysteine on each subunit (StvC; see PMID: 11716692) as described in our 2007 publication (PMID: 17187819). The plasmid for production of StvC is available from Addgene.
Most of the flow cytometry reagent manufacturers have spectra viewers available that include the reagents listed above. These include Becton Dickinson, Life Technologies (Invitrogen/Molecular Probes), BioLegend, and eBioscience.
Please contact us if you are interested in tetramers incorporating alternative, specialty labels. We may not be able to accommodate all requests, but we’d still like to assess demand.
Recommendations
We recommend using tetramers labeled with the brightest fluorophores that are consistent with the remainder of your staining panel. In most cases, this will be PE and APC, followed by BV421, and for class II tetramers, these are the only fluorophores that we recommend. The small molecule fluorophores —fluorescein and the Alexa dyes—are useful under the following circumstances: (1) when you want to stain with multiple tetramers; (2) when you are using protocols that require harsh denaturing conditions such as flow FISH; and (3) when you need the PE, APC, and BV421 channels for other dim or hard to obtain reagents.