Blocking: a cure for all your problems or a cause of all your problems?

In this series, I’m going to review blocking reagents and how to use them for best effect. I’m guessing some of you use blocking the way I did when I was starting out—as a cure-all for messy cytometry. Staining doesn’t look right? Add more block and more antibody. Binding to the wrong cell type? More block.

I think it’s important to actually test which reagents you need to use and how much you need. I say this because there are more and more blocking reagents available as we get more fluorophores and complex staining reagents, and you may not know that some of these reagents actually have detrimental effects. Hint: the more a reagent is hyped, the less you want to use it.

Some of these detrimental effects are open secrets in the field, but at least one that I'll describe is, I think, completely unknown.

So, in the next set of blog posts, I aim to cover the different types of blocking reagents, discussing what they’re meant to do, how well they do that, any side effects they have, and how best to use them. As always, these are opinions backed up with some data, but there will be cases that go against what I’m saying. Feel free to chime in with your experiences!

Some of the blocking reagents I hope to cover:

• Protein

• Fc block, IgG and serum

• Brilliant Stain buffer

• Tandem stabilizers

• Monocyte blockers

• CellBlox (AKA NovaBlock)

• Blocking for biotinylated reagents

For today, I’ll point out that titration is far more important than blocking. The affinity of the specific binding of your antibody to its target exceeds the off-target non-specific binding by orders of magnitude. That means that non-specific binding happens primarily due to excess antibody that can’t find antigen to bind to.

Here's an example using cryopreserved human PBMCs, gating on viable leukocytes.

Note the increased signal on monocytes (CD14+CD4-low) at the 1:20 dilution. The specific signal is better resolved at the lower dilution due to the decrease in non-specific binding. The cells here were stained for 1hr at room temperature, surface staining.

Reagents used:

CD14 Spark Blue 574

CD4 BUV395

NKp46 PE/Cy5

CD56 BUV563