For intracellular staining (such as for cytokines and transcription factors) in flow cytometry, we need to FIX & Permeablise the cells, prior the staining with fluorochrome-tagged antibodies. But by doing so, cells will loose its viability. Therefore, what is the benefit of doing live cell gating? Even some reviewer ask data for live cell gating. What does it mean?
Thanks
You would have stained for the dead cell prior to fixing the samples. That way, when you gate for the live cells, you only detect cells that were viable prior to fixing and permeabilizing.
Hello Mohammad,
The live cell gating allows to decrease significantly the "background noise" of non-specific attachement of fluorescent antibodies to dead cells. It is particularly important in the setting of identification of rare cells and functional studies (e.g. intracellular staining of cytokine-secreting Ag-specific T cells).
Indeed, you must stain the cells before the fixing and permeabilizing step. The best reagents for this are amine-reactive dyes, that are resistant to fixation and permeabilization.
They are now available from different commercial sources and with different fluorescent wavelengths (but may need carefull titration and compensation according to you panel).
The paper below may be helpful:
http://www.health.usf.edu/NR/rdonlyres/9CED577F-CB9C-4B92-99DB-A24B57F799F2/27244/aminereactivelive_deaddiscrimination.pdf
Hope this helps!
Marcelo
Hi Mohammad,
I agree with Marcelo, amine reactive stain works excellent! I use it all the time, fixed cells or not.
You can buy them from BD Bioscience, ThermoFisher Scientific, Biolegend or Affymetrix. Use cells for compensation or amine reactive comp beads from ThermoFisher or Bangs Lab.
Good Luck!
Kerstin
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