3 Choices to Make Before Intracellular Staining
Finding antibodies is the easy part of setting up your intracellular experiment. The difference between reproducible results or a do over is in choosing the right buffers, controls and concentration. Here are some of the finer points to consider when including intracellular staining as part of your experimental setup.
1. Choice of Buffer
Typically, cells are fixed and permeabilized in order to stain for intracellular targets. The location of the antigen of interest will be the deciding factor in choosing the appropriate buffer system.
Note: If you will be staining for nuclear proteins in conjunction with cytoplasmic proteins, determine if the antibody against the cytoplasmic protein is compatible with the Foxp3 buffer system. We have studied the compatibility of many cytokine antibodies in the Foxp3 buffer system and have listed the results here: http://www.ebioscience.com/resources/application/flow-cytometry/antibody-fixation-considerations.htm
2. Choice of Negative Controls
Negative controls are critical in analyzing your data, as many times there may not be a clearcut positive and negative population. Consideration of the various factors surrounding the use of negative controls will help you make the appropriate choices.
I. Isotype controls: We do not recommend the use of isotype controls for intracellular staining. Isotype controls can be a good place to start in setting up your instrument and controlling for the general autofluorescence from a conjugated antibody. Typically, one would expect that an isotype control would be negative in their staining, (i.e. very little shift over autofluorescence). But in fact many times the isotype controls do not perform as expected based on surface staining experiments. There are many reasons for this from inherent differences in the amino acid composition, to the different amount of fluorophore conjugated to the isotype control versus the experimental antibody. Hence, we recommend using unstimulated cells as a better negative control. (Fig. 2)
II. Unstimulated vs Stimulated: Whenever possible we recommend setting up samples in which you have added stimulant such as anti-CD3, CpG or LPS and compare these stimulated cells side by side with unstimulated samples (i.e. minus the stimulant). This setup allows for a more controlled experiment and is ideal for short stimulations (less than 24 hours). Running this control will allow you to better understand the expression of your protein. (Fig. 1)
III. Internal/Inherent Negative: Depending on your starting cell population and stimulation conditions, if any, utilization of a gating strategy based on the internal negative population (those cells known to be negative for the protein of interest) may be beneficial. For example, if you are stimulating cells in vitro to look at cytokine expression, you will end up with a heterogeneous population of cells; some expressing cytokine, some not. It is these non-expressing cells that can serve as the internal negative control.
The gating strategy above is based on the isotype control. If one were to use this gating strategy, there would be a high false percentage of cells staining positive (i.e. 31.91%). However, if you set your gates based on the internal negative population (IFNg negative - see red dotted line), you find the percentage drops to 14.19%.
It is important to note, if you are starting with an enriched population or cell line and are looking for a protein that is ubiquitously expressed (i.e. polarized cells stained for a transcription factor), this control may not be as useful because you have removed the internal negative population.
3. Choice of Concentration
Titrate, titrate, titrate. When performing intracellular staining, it is critical to titer the antibody for optimal staining of your cells. If you use too much antibody, you may start to see increased background staining which can lead to the entire cell population shifting and masking of the positive population. We have found that performing 2 fold dilutions of the antibody in order to determine the optimal concentration of antibody will yield the best signal to noise ratio.
We hope this information is useful to you and welcome questions and comments on this blog as well as any requests for future tech tips and protocols.