Blocking: Monocyte block

Today's post might be controversial. I welcome you to test the findings yourself. I have only used the BioLegend Monocyte Blocker and CellBlox, not BD MonoBlock.

The issue

Cyanine-tandem dye binding to monocytes and macrophages. This paper by Kristensen et al. nicely demonstrates the issue.

The reagent

BD MonoBlock

True-Stain Monocyte Blocker CellBlox

What is it?

Probably DNA. This paper, which first formally describes the effect, demonstrates that phosphorothioate oligodeoxynucleotides can block non-specific binding of Cy5 tandems to myeloid cells.

Alternatives

Sheared salmon sperm (DNA)

Mouse or human serum, but not commercial Fc Block, can accomplish similar effects, in my experience. This goes against the published literature.

What it’s supposed to do

Prevent/reduce binding of tandem fluorophores to monocytes. This is a definite issue for people working with human PBMCs.

Examples

I have trouble getting any beneficial effect on mouse cells, so here's an example using a PE-tandem on human CD14+ monocytes with surface staining:

Yes, it works. It doesn't completely block the non-specific binding, and the Fc blocking reagents perform better.

Here are some examples on human monocytes with overnight intracellular staining at too high a concentration of anti-CCR7:

Again, it works, but so do the Fc blocks. Monocyte blockers reduce non-specific binding of many conjugates, not just obviously cyanine-based tandems. In some cases this may be due to the incorporation of cyanine dyes as acceptor groups in the modern fluorophores (e.g., Brilliant dyes).

Any detrimental effects?

Oh yes! Very much so.

Naked DNA is not good for cells. In fact, neutrophils use it to capture and kill invading microbes. If you've ever isolated cells from tissues, you've probably used DNAse to reduce clumping, improve viability and improve yields. A lot of protocols use DNAse for cryopreserved PBMC thawing (it helps).

If you use Monocyte Blocker, the cells are much more likely to clump up, giving you fewer cells to work with.

Live leukocyte recovery with freshly isolated mouse splenocytes and cryopreserved human PBMCs (technical replicates). Data expressed as percent recovery, using the average recovery in FACS buffer (PBS 2.5% FCS 2mm EDTA) as the baseline.

Monocyte Blocker is not really any worse than CellBlox (if anything, the reverse). The difference is that Monocyte Blocker is being added as a pre-block while CellBlox (following the manufacturer's recommendation) is being added with the staining mix. Longer incubations give lower recovery. Generally, the effect manifests as an invisible loss of cells rather than a visible increase in dead cells; the loss tends to be higher among lymphocytes, skewing the cellular composition slightly.

Okay, so it's not great to use this on live cells. What about on fixed cells? Well, that gets rid of the problems of cell loss/killing, but we have a new issue.

When used intracellularly, Monocyte Block reduces transcription factor staining.

Examples with human PBMCs (overnight intracellular staining):

Examples with mouse splenocytes (overnight intracellular staining):

As far as I'm aware, this is a previously unknown effect.

Why is this happening? I don't know. I see two possibilities, assuming this is DNA-based: 1) Monocyte Block's DNA binds to histones, obscuring transcription factors that are bound to the cellular DNA. 2) Monocyte Block's DNA displaces cellular DNA in the transcription factor binding site, causing the transcription factor proteins to be released and float out of the cell--this is far-fetched because it would require a very broad set of DNA sequences (random oligos) in Monocyte block in order to displace disparate transcription factors.

How much to use?

The manufacturer-recommended test is 5ul per sample. The effects are concentration-dependent, and beneficial and detrimental aspects go hand-in-hand. To achieve complete blocking, you'll likely need 10-20ul per sample.

When do you really need it?

Well, as mentioned above, there is a beneficial effect with human monocytes. Perhaps if you're working exclusively on human monocytes and don't care about the lymphocytes?

Bottom line/how to get the best results

Don't use it? You can block non-specific binding of tandems to monocytes using serum (mouse, human, rat) and by titrating, in most cases.

If you do use it, minimize the time it's in contact with your cells. Add it to your staining mix rather than pre-blocking. CellBlox in particularly is suggested to be used in this manner to prevent the loss of cell viability/integrity. As I've communicated to the manufacturer, this doesn't entirely fix the problem.

Reagents used:

T-bet PE-Cy7

CCR4 PE/Fire 744

CD90.2 BV510

GATA-3 PE-eFluor610

TCF1 Real Blue 705

FoxP3 Alexa Fluor 647 Foxp3 PE

CD3 Spark Blue 550 T-bet PE/Dazzle 594 CD56 BUV563

TCR alpha/beta APC