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|
|BV421||405||421||Violet||Bright replacement for PacBlue|
|BB515||490||515||488||Bright replacement for FITC, Alexa 488|
|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.|
Streptavidin-PE and APC are currently obtained from Agilent. Streptavidin-Brilliant Violet 421 (BV421) and Brilliant Blue 515 (BB515) are obtained from BD Biosciences. Streptavidins labeled with the small molecule fluorophores (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.
With the addition of BB515 we will no longer offer FITC and Alexa 488 tetramers. If you have experiments in progress with these fluorochromes or have other reasons to request these we can make accommodations, please contact us directly about this. See the figure below for a comparison of FITC, Alexa 488 and BB515 labeled human MR1 tetramers used at the shown dilutions to stain human peripheral blood. Plots show CD3+CD19- lymphocytes.
Several online spectra viewers are available that include the reagents listed above.
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.
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 BB515. For class II and mouse MR1 tetramers we especially recommend PE and APC; BV421 and BB515 are also available but usually don’t work quite as well. The small molecule fluorophores (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, BV421, and BB515 channels for other dim or hard to obtain reagents.