Combine Immunofluorescence and flow cytometry

Analysis of protein expression remains a central procedure in life science research. Detection of protein expression with antibodies can use a variety of platforms like ELISA, Western Blotting, Immunofluorescence, Flow cytometry and Immunohistochemistry. Biologists are used to testing in one of these platforms at a time. This is because each of the procedure usually takes up all of the experimental material (cells) OR all the time (a workday or two) while the live cells are available. Additionally, many scientists like to focus on one experiment at a time, to minimize the possibility of introducing handling errors. This is a sound approach since it is just as important to obtain good data with one technique as it is to validate that data in another technique. But this approach increases the cost of research in the form of the harvested biological material and the time that is invested in repeating the experiment in another platform another day.

While it may not be possible to combine Western blot, ELISA and immunohistochemistry, the remaining two techniques offer a possibility of combining into one experiment. Both Flow cytometry and Immunofluorescence involve labeling the cells with fluorescent primary or secondary antibodies and detecting the immunofluorescence signal. This requires a simple tweak in the workflow: stain the cells as if you were simply doing a flow experiment. Then split the sample just before analyzing on the FACS machine, and transfer one fraction of the stained cells onto Shi-fix™ coverslips. Allow the stained cells to bind to the coverslip for 30 mins, carry out a quick nuclear counterstain with DAPI for 5 mins, wash and load the coverslip onto a slide. Now you can obtain flow cytometry AND immunofluorescence data from the same day-long experiment! This also works for surface stained OR PFA-fixed triton-permeabilized cells, so do not hesitate to give it a try !!