At 2:16 PM -0800 3/16/00, Mario Roederer wrote: >Yes, we do this routinely using Ethidium Monoazide (EMA). EMA works >like PI, with the bonus that when you expose the cells to light, the >EMA covalently links to the DNA. Thus, the protocol is to incubate >cells with EMA much like PI (in the dark) during the time when they >are still alive, perhaps during staining, then wash them, then >expose them to light for 10 minutes or so. Then you can fix & perm >and do whatever you want to them. > >The only downside is that you must devote a whole channel to the EMA >measurement (usually, FL3). EMA is not as bright as PI, so you >can't overlay an immunofluorescence stain on top of the live/dead >measurement as you can with PI. > >Although the EMA protocol was first published about 10 years ago, >you can access our more recent protocol, where we optimized it for >use with infectious PBMC & intracellular staining after stimulation: > >Mitra et al (1999). Differential representations of memory T cell >subsets are characteristic of polarized immunity in Leprosy and >atopic diseases. Intl. Immunol. 11:1801-1810. > >mr I want to add that we now routinely use Mario's method and it works very well and is easy to do. It helps enormously in identifying rare cells when using fixation. We have used it in conjunction with BrdU detection, TUNEL FACS and intracellular cytokine detection. Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 203-688-2748 (fax) mark.shlomchik@yale.edu
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