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.