Recently, we discussed Monocyte blockers. CellBlox is a special case because it was originally designed for a different reason: to block non-specific binding of NovaFluors, both to cells and to each other. It also happens to work well to block cyanine dye binding to monocytes.
The issue
NovaFluors are DNA origami structures (Phitons) that allow for specific placement of fluorophore FRET partners via oligo hybridization. These are very large molecules, much bigger than antibodies. The naked DNA can be bound by cells via receptors designed to recognize and internalize foreign DNA. If you're working with immune cells, as most of us are, this is a problem. DNA also binds to dead cells, perhaps via histones, although it may just be because these are large molecules with many sites for potential interaction. The DNA-based fluorophores also interact with each other, like Brilliant dyes do, but much more so.
So, there is undesired binding to cells, dead cells and between antibody conjugates.
The reagent
CellBlox and the new, improved CellBloxPlus (the one I'm using here). Previously known as NovaBlock.
What is it?
Unclear, but I would guess it's the Phiton DNA origami structure without any fluorophore added. CellBloxPlus may be a more concentrated version.
Alternatives
If you're after a monocyte block, you have other options.
To block NovaFluor interactions or undesired binding to cells, I have found no alternative that works nearly as well. DNA, such as Sheared salmon sperm (DNA), can reduce binding to dead cells, but doesn't touch the interactions between NovaFluor conjugates.
As noted in OMIP-102, some of the non-specific binding is due to aggregate formation and can be mitigated by centrifuging the product at high RCF prior to use. With any product that forms aggregates and requires a hard spin before use, you will reach a point where there is unusable product (most aggregates) left in the vial. Knowing when you have reached that point is a gamble. Personally, I dislike using any reagent that has the potential to cause a large, variable amount of non-specific staining simply by using it up, but this is a post about CellBlox, not NovaFluors.
What it’s supposed to do
Allow you to use NovaFluors and obtain specific staining. With most NovaFluor conjugates, you cannot get away with not using CellBlox.
Examples
Most of the NovaFluor conjugates I have target murine antigens. With mouse cells, blocking cyanine dye interactions with monocyte blockers isn't very effective, as we see below. There's a spike of non-specific CD25-PE-Cy5.5 staining correlating with F4/80-PE-Cy7. This doesn't go away when CellBlox is used. In fact, there's a large increase in PE-Cy5.5 binding in correlation with the CD19-NY690.
C57BL/6 mouse splenocytes stained for 1hr at 4C (surface staining).
How about the non-specific binding to cells? Well, I didn't spin these conjugates prior to using them, and they're 3-4 years old now since I stopped buying these products, so it's possible they're worse than they were initially. In the example above, CellBlox hasn't prevented non-specific binding of anti-CD19 to most of the cells, as evidenced by the large plume of CD25 (Tregs) being marked as CD19+. Personally, I find that you need a bit more than the recommended amount to block the non-specific binding entirely.
In some cases, the benefit is still measurable. Here we see that CellBlox has reduced the interaction between anti-CD4-NB610 and anti-CD19-NY690. Note that Fc block does nothing to fix this.
In the example below, we can see some improvement in the specificity of anti-CD19-NY690, and a reduction in CD8-NB610 when CellBlox is used.
On dead cells, CellBlox definitely helps non-specific binding, which isn't really replicated by Monocyte Blocker or Fc block. The dead cell binding can be blocked with random DNA, like the sheared salmon sperm mentioned above. While it isn't super critical to remove non-specific binding to dead cells from an analysis point of view (the first step is to exclude these cells), if all of the conjugate is going towards binding to dead cells, there won't be as much left to bind to our desired target, and we'll get worse staining.
Any detrimental effects?
Yes, definitely. As recounted in the post on monocyte blockers, CellBlox reduces cell recovery. When used according to the new recommendations (adding to your staining mix rather than pre-incubating with your cells), the loss of cells is moderate and probably manageable for most users.
This is what happens if you pre-incubate:
Anything else? Yes, there are sometimes really odd effects on the staining. I would guess that this has to do with non-specific binding to cells that have been damaged and lost membrane integrity, but I don't know. Example below on human cryopreserved PBMCs.
Note that we saw a similar effect on mouse splenocytes above, where the CD19 vs CD25 profile shifted radically.
How much to use?
The effect on cell recovery is dose-dependent, so if you can reduce the amount of CellBlox required, you'll minimize this. Using only 1 or 2ul instead of 5ul can reduce the cell loss to negligible amounts. In my hands, though, I found I needed more like 10ul to actually block all the undesired staining. So, test and find out.
Interestingly, I've observed that the non-specific binding of NovaFluors can become less of an issue with preparations of cells from tissues. I would guess that the higher level of debris in these samples (and probably DNA/nucleotides) can absorb/block some of the undesirable interactions. So, having dirtier cell preparations may be beneficial.
When do you really need it?
When using NovaFluor conjugates, particularly if using multiple conjugates.
Add it to your staining mix (like Brilliant Stain buffer), rather than adding to your cells like Fc block.
Bottom line/how to get the best results
If you are using NovaFluors, you absolutely need to use CellBlox, and should be using CellBlox Plus.
If you aren't using NovaFluors, the disadvantages likely outweigh any benefits.
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