Frequently Asked Questions
1. What should I do if my experiment fails?
2. Is there a model system I can try first before my own experiment?
3. What if I can't see any differences between my stimulated and unstimulated samples?
4. What controls should I have?
5. How do I enhance the staining (i.e. create a larger shift between my stimulated and unstimulated samples)?
6. What should I do if I have high background?
7. What is the optimal number of cells to stimulate?
8. Do I have to permeabilize with methanol?
1. What should I do if my experiment fails?
If the experiment fails on the first attempt, try again. These protocols must be followed. They are relatively simple, but deviation from them can and will cause an experiment to fail.
2. Is there a model system I can try first before my own experiment?
Yes. We highly recommend performing the Sample Experiment #1: Human Cell Line. It is consistent and gives high fold changes when analyzed with FACS.
3. What if I can’t see any differences between my stimulated and unstimulated samples?
- Make sure that the stimulation conditions induce the proper phosphorylation by Western blot. Then re-attempt the FACS experiment.
- Re-titrate the antibody.
- Make sure that unstained cells appear on the scale of the FACS plot.
- Often the staining intensities are low and may be missed if PMT settings are too low.
4. What controls should I have?
Be sure to have control samples of unstimulated cells. Also, cells that have been fixed and permeabilized but stained with an isotype control or left unstained are useful.
5. How do I enhance the staining (i.e. create a larger shift between my stimulated and unstimulated samples)?
- Optimize fixation time with formaldehyde
- Permeabilize with methanol for 10 min
- Optimize antibody staining time and titration
6. What should I do if I have high background?
Make sure there is a blocking agent in the staining media such as BSA. If necessary, block with non-specific mouse IgG (for monoclonal Abs) or rabbit IgG (for polyclonal Abs).
7. What is the optimal number of cells to stimulate?
Stimulate a large number of cells to ensure good titrations. Stimulating 1 million cells per sample is the easiest and most consistent. One can stimulate fewer cells, but titrations become more difficult as relatively more cells are lost during fixation and permeabilization.
8. Do I have to permeabilize with methanol?
Methanol is most advantageous for phospho-epitope analysis. However, saponin is also an effective permeabilizing agent for some phospho-epitopes. Please see Publications for more information.
