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Fixative stability – can your dyes do this?

Can your tandem dye do this?  

Well, no, it cannot.  As anyone who has attempted to ride a tandem bike can attest, it seems like a good idea but puts a serious strain on the relationship.  While that might be an overshare, dyes are no different — when attached to one another in a tandem dye, they break down over time in solution and break down in hours in fixative solution (Maecker, H.T. et. al. Cytometry A. 2004.; Tario & Wallace. “Reagents and Cell Staining for Immunophenotyping by Flow Cytometry” 2014.)

Really?  Yes, really.  

Sorry but I have some more bad news about tandem dyes. They are a pain to compensate, specifically:

  • You must use the same antibody conjugate to conjugate as you are using in your staining: this makes compensation of dim markers or those staining low frequency populations difficult — requiring beads in many cases.  
  • You should not compensate using a vial of the same antibody-tandem conjugate if it was just purchased just a month later (even if is the same lot), as degradation of the tandem can occur. 
  • Also, its best practice to compensate your tandem-dye-antibody conjugate with a fresh source rather than a vial from the same lot that is month old. (“Tandem Fluorochrome Conjugated Antibody Best Practices.”  University of Iowa. https://medicine.uiowa.edu/flowcytometry/protocolssample-prep/sample-preparation-analysis/tandem-fluorochrome-conjugated-antibody-best). 

Really?  Yes, really.  

The perfect fluorescent label would be fixative stable and be stable across the entire spectrum*.  

*It should also be easy to compensate.  Okay, you’re on. 

  1. NovaFluor conjugated antibodies are stable on stained cells in fixative solution for at least 2 weeks (2% paraformaldehyde shown above).  …and I don’t mean let’s fix-and-resuspend-in-PBS stable.  I mean full tandem-destroying 2% paraformaldehyde for 2 weeks. 
  2. Moreover, they are stable across the entire spectrum for that time (see data above from our Attune).  We are running follow-up experiments now on spectral instrumentation as well.
  3. Compensation?  No problem, you can compensate any NovaFluor on any marker e.g. CD4, CD8, whatever.  There is no change in fluorescence when they are conjugated, unlike Tandems. 

In the end, NovaFluors and Tandem dyes are not on the same wavelength.  As John Keats who claimed that “There is nothing stable in the world; uproar’s your only music.”  Let’s leave the chaos to the beat of the butterfly’s wings, and replace your tandem-conjugated antibodies today with stable, consistent NovaFluor-conjugated antibodies. 

We also now offer 25 test versions of all our antibodies!  Now that’s a combination worth considering. 

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