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3 ways to maximize your S/N ratio

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Maximizing the signal to noise (S/N) in a flow cytometry experiment is the goal of all researchers.  

It may be easy to detect a marker like CD3 on T cells, but to measure rare, dim or emerging makers requires a good S/N. There is no one magic way to improve the S/N – rather the enemies of a good S/N must be confronted head-on and addressed with proper experimental design.

Here are 3 ways to maximize your S/N ratio:

1. The Problem:  High Antibody Concentration  

If an antibody cannot bind to the primary target, it will bind to low affinity targets. The non-specific binding increases background and thus reduces the S/N of the antibody. This is especially true when targeting intracellular proteins.  

The Solution:  Titration 

Performing a titration experiment will allow the researcher to identify the optimal concentration of antibody to be used for an experiment. As shown below, outside of the boxed region, the S/N decreases because either there is too little antibody (left of boxed region) or too much antibody (right of box region).  

2. The Problem:  Dead Cells  

Dead cells will non-specifically uptake all the antibodies in the mix. These antibodies enter the cell and bind to the low-affinity targets inside the cell (e.g. cell matrix), masquerading as live cells.

The Solution: Viability Dyes 

Elimination of dead cells using a viability dye will eliminate these false positives. If the cells are not fixed, then a cell impermeant dye like PI, 7AAD or DAPI can be used. These cannot enter cells with intact membranes, and are colorless until bound to DNA. If performing an intracellular stain, then consider using the various Amine reactive dyes. These dyes work by binding the amine groups present on proteins. Dead cells will bind much more of these dyes, identifying the cells that were dead at the start of the experiment.  

3. The Problem:  Fc Receptor Binding 

The Fc receptor (such as CD16 and CD32) are found on the surface of many different immune cells. The role of the FcR is to bind the constant region of an antibody that has bound to a target identified for elimination. The Fc receptor is a specific binding that can reduce the S/N by binding all the antibodies in the staining mixture.

The Solution:  Proper Blocking 

Blocking of the cells before labeling with antibodies will reduce this problem. One can use specific FcR antibodies, but those can interfere with downstream assays. It is often better to use 5-10% normal serum of the animal that your target antibodies were raised in (i.e. use normal mouse serum for mouse Ab’s). Incubate the cells for 10 minutes before labeling is all it usually takes to block the FcR binding. 

Keeping in mind these three solutions will help ensure that the causes of reduced S/N are addressed in the experimental design and protocol execution.