Experimental Tips
There are several important factors to consider when attempting to apply phospho-specific antibodies to flow cytometry.
- Stimulation conditions must be found that strongly induce the phosphorylation event of interest. For example, PMA induction of phospho-ERK.
- The antibody must be checked by Western blot to ensure specificity and selectivity for the protein of interest. An antibody that recognizes many bands by Western will not prove very useful for flow cytometry (or immunofluorescence) because it will have a high background staining intensity.
- The antibody must be conjugated with a fluorophore, such as FITC or one of the Alexa Fluor dyes (Molecular Probes), in order to perform multi-parameter experiments. Secondary antibodies can be used in pilot experiments to titrate the phospho-antibody or to determine if the antibody will be effective.
- One must freeze the signaling events within the cells rapidly and efficiently. Formaldehyde fixation appears to be an effective means of halting signaling cascades for subsequent permeabilization. Percentage and time of formaldehyde fixation can be changed to optimize the signal-to-noise ratio.
- Conditions must be found that efficiently permeabilize the cells of interest and provide an accessible epitope for the phospho-antibody. Many methods are available to permeabilize cells including saponin (0.1 to 0.5%), Triton X-100 (0.1 to 0.5%) or alcohols like methanol and ethanol (70 to 100%). Different conditions may benefit particular epitopes. However, methanol appears to work generally for most of the phospho-epitopes we have looked at.
- The antibody must be titrated on stimulated vs. unstimulated cells to find the optimal concentration that maximizes the difference between median fluorescence intensities (MFIs) of the two samples. In general, low concentrations of antibody provide the best staining, such that unstimulated samples appear close to isotype control MFI.
- Staining times may need to be adjusted for particular epitopes. In general, 30 minute staining times are adequate.
- The cells can be kept at -80°C for up to a month in methanol and still be adequately stained. However, we have not conducted a thorough study of this as yet, and do not guarantee that every cell type and condition will stain successfully.
- All of these parameters are important to experimental success. Small changes to the protocol outlined on this website can make significant changes to the quantitative outcomes of the experiment, i.e. of 30% or more. Therefore, care should be taken to be experimentally consistent between samples and between days of experiments.
