I am trying to develop a method to stain suspension ES cells, which will be later quantified using flow cytometry. However, the result I get from flow cytometry shows that only a small population of cells is stained. The result doesn't match with our previous result, and previous result shows that almost 100% of adherent ES cells are stained (base on microscopic observation). So I suspect if we need a higher grade of antibody, which is suitable for flow cytometry, or the same antibody which is applicable to adherent cells and also suspension cells?
You can't just use the same antibodies for flow cytometry and immunohistochemistry. First you should consider to use fluorochromes and antibodies for immunofluorescence and check on the datasheet if the same antibody could be use for both the techniques. Second, fluorochromes for flow cytometry are designed both to cover a specific wavelenght range and to have specific dimensions (which may not be suitable for both techniques). Third, I am not sure but I don't think aderence to a surface affects antibody hybridization on the cell (in facts ES in suspension should be marked more efficiently) but, since ES often require adesion and feeder cells support, they may not be in their optimal condition and therefore not express all the markers you are trying to mark. As a solution you could try and use the Aldefluor marking technique (which is not based on antibodies) which is useful to mark stem cells in general both in vitro (only viable cells) and in flow cytometry.
Each antibody has to be validated for every technique in which you want to use them. You need to have a negative control and a positive control and then test different concentrations/dilutions - the optimal concentration/dilution may not be the same for FACS and IHC. And some antibodies may be good for IHC but not FACS. In your case, if you increase the concentration of antibody to detect 100% positive ES cells, make sure that that same concentration still yield 0% in your negative control cells.
If you use an antibody that has been use by a number of other research centres (check their publications), you increase your chances to get a good result in a minimal amount of time. Spend some time researching these, you will save a lot of time on the testing and a lot money on crappy antibodies !
Good luck!
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