Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.
Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin. Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light-scattering properties.
Riedy
M C
MC
Roswell Park Memorial Institute, Department of Flow Cytometry, Buffalo, New York 14263.
Muirhead
K A
KA
Jensen
C P
CP
Stewart
C C
CC
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001386:Azides; D002470:Cell Survival; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D016130:Immunophenotyping; D007834:Lasers; D013048:Specimen Handling
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001386:Azides; D005404:Fixatives; D005456:Fluorescent Dyes; C014535:8-azidoethidium