Optimisation of blocking during flow cytometry

Another new flow cytometery guide drop from Oliver Burton, just published in Current Protocols.

This one is on optimising blocking while preserving signal, in particular how to overcome Fc interactions and dye-dye interactions, and preventing tandem break-down.

The details are in the protocol, with variants for intracellluar and cytokine staining, but generally-speaking normal mouse/rat serum, BioLegend Tandem stabilizer and Thermo/BD Brilliant Stain Buffer is an optimal combo. True-Stain and other additions aren't worth the extra $$$.

For Tandem Signal Enhancers, you don't really need them for mouse cells, and for human cells a cheaper alternative is simply to fix your cells and stain with tandems after fixation. Both eliminates non-specific tandem binding and also reduces tandem break-down. Since monocyte blocks reduce transcription factor detection, for some reason, leave them out if you are doing intracellular staining.

 

The Brilliant and Super Bright dyes really do need the Brilliant Stain buffers, but be aware that these buffers are mildly fluorescent, so leave them out if you don't need the dye, and titrate them down when you use them. 1/2 to 1/4 is normally good, and for many antibodies even lower is fine (and cheaper!).

For the tandem dyes, Tandem Stabilizer is good, but you can make it easier through panel design. Tandem breakdown is not purely chemical - it is higher on monocytes than lymphocytes, and is largely abolished in fixed cells. So move those tandem dyes to post-fix T cells if you can!

 

We've tried to cover all the main use cases, so take a look at Oliver's trouble-shooting guide to reduce off-target binding and preserve signal.

A near-universal ultra-cheap fix/perm protocol for flow cytometry

For our flow cytometry peeps, would you like to have a single fix/perm protocol that is optimised for everything? One that preserves fluorophores while allowing simultaneous TF and cytokine staining? How about a protocol that is 100-fold cheaper than your current one?

Over the last 8 years, Oliver Burton has tested >1000 different fix/perm combos, and here the final verdict is: "Burton's Best Buffer": 2% formalin, 0.05% Fairy dish soap, 0.5% Tween-20, 0.1% Triton X-100.

Yep, replace all of those expensive detergents with Fairy dishwashing liquid. It is as good as the BD Foxp3 fix/perm kit for transcription factors, as good as eBio perm for cytokines, preserves even weak endogenous GFP killed by most fix/perm combos, and preserves dye integrity too. Burton's Best Buffer is simply the best fix/perm protocol to use under any condition (except phospho-flow).

Plus it is dirt cheap - one bottle of Fairy (or Dreft, Dawn, Yes, JAR, or whatever they sell it as locally) will literally last your lab for decades.

Take a read of the protocol here.

Overnight staining of flow cytometry samples │ Oliver Burton