Is it possible to use multiple stains on same cell line and analyze them by flowcytometry?

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Nicolas Tremblay

Hi Zack,

this is the essence of flow cytometry. You can use many compatible fluorescent-labelled antibodies to label surface or internal cell markers. FACS enables you to measure the fluorescence of the dyes witch is ''proportional'' to the presence of your protein of interest. This enables you to differentiate between cell populations and, thus, to test out your research hypothesis.

You probably have a flow cytometry core facility or an expert lab nearby. They would be of great help to get you started!

You can watch this intro video to learn more about FACS: https://www.youtube.com/watch?v=UcjhdjsOG1E (skip to about 6:10 for antibodies)

And this video for an applied example of multi-color FACS: https://www.youtube.com/watch?v=BIgChI_UfAc

Zack Hi

Thank you Nicholas,

I think I forgot to mention what I'm really staining. I want to do cell cycle analysis and at the same time to test for surface receptor. Will there be a problem if add the antibody for surface receptor and then fixate the cell cycle analysis?

Andrew Wight

Hi Zack - yes, you can analyze surface and intracellular markers in the same experiment, although it takes a bit more work and you have to do at least two rounds of staining. I've linked two protocols to give you a starting point: the first is a protocol from Abcam describing how to do cell cycle analysis using propidium iodide, which mentions how to do surface stains followed by PI staining. The second is a more step-by-step description from eBioscience. Good luck!

http://www.abcam.com/protocols/flow-cytometric-analysis-of-cell-cycle-with-propidium-iodide-dna-staining

http://www.ebioscience.com/media/pdf/best-protocols/staining-intracellular-antigens-for-flow-cytometry.pdf

Thank you Dr. Wight, that was helpful.

Now, after my staining analysis is done, do I have to remove the stain or it is OK to keep it?

Jitendra Kumar Shandilya

this is the beauty of flowcytometry dear.

By flowcytometry you can actually analyse the co-expression of multiple cell markers on a single cell. number of markers to be analysed depends on the filters available in your flowcytometer, that may vary from 2 color to 17 colors.