Live/Dead Dyes: Why They Should be Included in Your Panel
|A successful experiment is surely a very happy event for anyone working at the bench. While it doesn't mean that the data is going straight to a scientific journal, it is definitely the building block to completing your thesis or project. Even if you get negative results, it is important to know that you can trust your data.|
|In that sense, it is widely recognized that the quality of your sample is directly reflected in the quality of your data. In flow cytometry, as in any other technique, it is important to ensure good processing of your sample. There are specialized tools for flow cytometry to monitor the number of dead cells and amount of tissue debris present.
Certain cell types (e.g., bone marrow resident plasma cells, mouse intestinal lamina propia cells) are more sensitive than others, and lengthy manipulation and protocols can affect the viability of your samples. In these cases, it is particularly important to exclude dead cells from your analysis.
When someone interrupts me while I am processing that Nobel prize-winning sample.
|In addition, it is well known that dead and apoptotic cells can capture antibodies non-specifically. Thus, exclusion of these cells is a common practice for an accurate representation of your populations. This is even more important when working with small populations or rare events, as even small changes can have a big impact in your calculations.|
|As shown in this example, the percentage of CD45 and CD11b positive cells change depending on whether the gated cells are dead or alive.|
Viability of mouse tumor samples was assessed with DAPI, cells were co-stained with the markers shown. Kuonen, F et al. 2010. Cytometry. 77:1082.
|Dead/live dyes in general work by binding to molecules not normally accessible to them in healthy cells. These molecules are exposed when the cell dies and the membrane becomes permeable. For example, 7-AAD and Propidium Iodine can intercalate into the DNA. Other dyes react with amines (present in proteins). In a healthy cell, they can only bind to amines on the surface. Once a dying cell becomes permeable to the dye, they can also react with all the intracellular amines, resulting in increased intensity.|
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