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Antibody Titration-The Key to Customizing your Panel Design

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Posted by FlowMetric on: March 17, 2015

We’ve talked about the three key elements you need to design an optimal panel for your next flow cytometry experiment: 1. Know your equipment, 2. know your species, and 3. know your fluorochrome. Now you might be ready to stain some cells, run them through the cytometer, and acquire some perfectly usable data.

Not so fast! Your newly arrived fluorochrome-conjugated antibodies probably came with some general instructions, which included some beautiful histogram plots showing how well the antibody stained cells carrying that particular target. This doesn’t mean your particular cell population will look anything like this when you blindly use the staining protocol recommended by the manufacturer.

Now is the time to do that “pre-experiment” type of experiment and titrate your antibody using cells and conditions that most closely mimic your experimental conditions.

You will soon observe that the overall brightness of each fluorochrome varies. Some are much brighter than others, and this can affect how your fluorochromes work on a particular instrument or with your cell type. You may also discover you can use a lot less antibody (and save some precious lab $$) for each experiment.

Thinking about Titration 

Experienced flow cytometry users consider antibody titration a critical step for optimal panel design. Tony Chadderton, Senior Project Manager at FlowMetric, Inc. said, “Titrations are important to make sure that the fluorescence intensity of antibody stained cells is linearly proportional to the protein being measured and that the signal to noise ratio of the fluorescence for the antibody is maximum.”

So now that you know you need to run a titration, think about these elements of your experiment:

  1. Are you staining a target that is expected to be highly expressed or weakly expressed?           

You’ll want to titrate your antibody using the same or similar cell type as your plan to use in your experiments.

You may also consider using a cell type with maximal expression of your target so you can pinpoint the antibody concentration that can stain an abundant amount of target without causing too much non-specific background staining.

      2.  Is your target expressed on the surface or is it intracellular?

You’ll want to use the same staining process for your titration as you’ll use for your experiment, and this differs for surface or intracellular targets.

      3.  Do I need to treat my cells with something to block non-specific binding of my staining antibody?

Different antibody isotypes can bind non-specifically to Fc receptors on the cell surface. Check out the protocols provided with your antibody or in the methods section of scientific manuscripts that use this antibody to avoid this potential pitfall.

Doing the Dilutions

Now it’s time to set up your titration. You’ll want to use the same conditions as much as possible in your titration as you plan to use in your experiments. This means using the same number of cells in the same volume of liquid, the same buffers, the same number of wash steps—all those details are important. You’ll probably want to start with a high concentration of antibody (like 1 mg antibody/100 ml sample) and make a series of 8 to 10 3-fold dilutions to get a broad range of antibody concentrations. As you set up your dilutions, you’ll want to remember to reserve a sample with no antibody for use as unstained control cells. After you dilute your antibody, you can add your cells, incubate and wash them according to your experimental protocol, and read them on your cytometer.

So What Am I Looking For?

Now that you have run your titrated samples, you’ll need to calculate the stain index to determine what that optimal antibody concentration is for your experiment. You’ll notice that there will be both stained (positive) and unstained (negative) cells at each antibody concentration. Once you’ve acquired this data, you’ll need to analyze each stained sample to determine the standard deviation (SDnegative) of the negative population (SDnegative) and the median fluorescence intensity (MFI) of the positive and negative populations.

The stain index can be calculated for each antibody concentration using this formula:

SI = (MFIpositive – MFInegative)/(2 x SDnegative)

Next, you can create a scatter plot with the log antibody concentration on the x-axis and the stain index on the y-axis. The peak of the curve generated by the scatter plot is the optimal antibody concentration.

Stay tuned for our final blog in the series, "Know your Flow", highlighting Step 5: Panel Optimization…