PI and 7-AAD in particular have a long history in flow cytometry applications. Examples of these dyes include the Sytox dyes, DRAQ7, propidium iodide (PI), and 7-aminoactinomycin D (7-AAD). They are the first type of live dead cell dyes that most scientists and flow cytometrists consider for their experiments. Classic DNA dyes.Ĭlassic DNA dyes are exactly that-classic. Adding the right reagent will result in increased quality in your data and increased confidence of your conclusions by both you and those reviewing your grants and papers. With the following three options of live dead cell reagents available to every scientist and flow cytometrist, you should have little trouble finding a dye that fits into your antibody panel and your flow cytometry assay conditions overall. These methods can be divided into three reagent classes, including classic DNA dyes, amine reactive dyes, and vital dyes. There are several methods for analyzing live, dead, and apoptotic cells by flow cytometry. 3 Dead Cell Reagents To Improve Your Data Analysis Finally, by adding anti-CD45 antibody to the sample (right panel)you see both the autofluorescent population, as well as a separate CD45-positive population that’s taking up the antibody.įortunately for scientists and flow cytometrists like you, there are multiple ways to label and identify dead cells so they can be removed from your flow cytometry analysis and cell sorting experiments. Also, as discussed above, the left panel reveals the higher levels of autofluorescence in the dead cells. This would NOT be possible without the dead cell marker. Here, you can easily see what appears to be CD45 contamination of their MSC product in the dead cell (dead cell marker-positive) fraction. In the panel on the left, the sample is stained with a dead cell marker only. The lab in question is very good at producing these cells from bone marrow and routinely generates 100% CD45-negative cells after their analysis. The two plots display mesenchymal stromal cells (MSC) produced in a Good Manufacturing Practices lab. Why You Need To Remove Dead Cellsĭead cells should be removed from all flow cytometry experiments that aim to evaluate live cell lineage and functionality.īelow are two simple examples of why you need to remove non-viable cells prior to implementing your flow cytometry gating strategies. These last two issues, in particular, will become even more troublesome if you are using dim markers or rare cell types. To make matters worse, both dead cells and apoptotic cells are highly autofluorescent. If you failed to remove your dead cells beforehand, gating on your population of interest will be exceptionally difficult. The problem here is that dead cells take up antibody very readily. In one final example, let’s say you’re simply staining surface antigens in a population of cells. This means you will end up with fewer vital cells in one set of wells versus another, altering your results and negatively influencing your interpretation of the data. If you failed to remove your dead cells first, you could end up with different percentages of dead cells in one sorted sample versus another sample. Or maybe you’re sorting cells for a downstream functional assay, stimulating sorted T cells with antigen and measuring production of IFN. Since the dead cells in your sample will not divide, your culture will take extra time to reach the needed level of confluence, ruining your experimental timeline and weekend plans. If you fail to remove your dead cells first, you might think you’re seeding 10,000 cells, but in reality only 7,000 of your cells are actually viable. For this and other reasons, it’s important to remove dead cells from further analysis during your flow cytometry experiments.įor example, let’s say you merely need to generate an accurate cell count. This allows for antibodies to penetrate the cells, which can now mimic live cells. As cells die, the membrane becomes permeable.
0 Comments
Leave a Reply. |