Measurement cell death pdf

Now some researchers are concerned about adding these dyes to a sample in case they will impact downstream analysis, especially in Genomics. This is not the case for two reasons. First, the amount of dye used for this purpose is much lower X than used for cell cycle analysis. Second is the dilution factor. If you sort a cell with an micron nozzle, the droplet size has a volume measured in nanoliters and the core stream is only a fraction of that volume, so the amount of the dye that will be remaining after sorting is negligible.

Since these dyes require an intact membrane to exclude the dye. There have been various techniques used in the past, to varying degrees of success. With the introduction of the amine reactive dyes, the ability to identify dead cells became that much easier.

These dyes work by binding to the amine groups on proteins. As shown in Figure 1 above, these dyes will bind to the surface of a cell, making live cells slightly positive. Dead cells, with compromised membranes, allow for the dye to enter the cell, where there are a lot more proteins for the dyes to bind to.

Figure 3 : Identification of dead cells using the amine reactive dyes left. The figure on the right is from the Thermo Fisher website showing the staining of live and dead cells.

This can be a damaged cell, or it can be part of the normal biological process of development. For example, in utero the fingers are webbed and it is through ordered apoptosis that this webbing gets removed. In Apoptosis, one of the earliest signals is the flipping of the phosphatidylserines, which face the cytosolic side when a cell is living, but by the action of flippase, face the extracellular milieu when a cell is undergoing Apoptosis.

Annexin V is a calcium-dependent phosphatidylserine preferentially-binding protein. When coupled with a cell impermeant dye, it is possible to dissect the stages of apoptosis, as shown in the figure below. Figure 4 : Annexin V staining of cells undergoing drug treatment.

Cells in the lower right quadrant bind Annexin and are at the early stages of cell death. The Annexin V assay lends itself to high content screening assays, which makes it ideal for monitoring cell death in drug screening assays. Another assay that can be run to look for the earliest stages of apoptosis uses two different dyes. Using these two dyes, it is possible to identify the apoptotic cells from necrotic cells. Necrosis is disordered cell death, usually due to traumatic cell damage and the release of proteins from the lysosomes a process called autolysis.

The example data, courtesy of Derek Davies, shows an example of how these two dyes work. Figure 5 : Measuring apoptosis and necrosis by flow cytometry. Data courtesy of Derek Davies. Another hallmark of apoptosis is the depolarization of the mitochondria and the release of cytochrome C.

Cytochrome C can be measured by intracellular staining using an anti-Cytochrome C antibody. As Cytochrome C is released, the amount of staining will go down. Example data from King et al.

In this assay, cells were treated with staurosporine to induce cell death. The process of Cytochrome C release requires the depolarization of the mitochondria. On the left are the untreated cells, while on the right are the treated cells. The depolarisation shows a decrease in the signal of the CMXRos.

Data from Derek Davies. Cell death is a normal biological process that is amenable to measurement by flow cytometry. Cell impermeant dyes are an absolute requirement for cell sorting experiments, or for live cell analysis. Since dead cells can mimic the target cell, these must be eliminated. For intracellular staining, the amine reactive dyes are an excellent choice.

Annexin V assay is a good assay to measure cell death and is a great tool for looking at cell death in a high-throughput assay. Additionally, there are several DNA dyes that can be used in this process as well. Finally, measuring mitochondrial depolarization is accomplished with several different dyes, resulting in a different measurement for cell death.

Cohen, G. Fadok, V. Rodriguez-Tarduchy, G. EMBO J. Lennon, S. Cell Prolif. Martin, S. Hickman, J. Cancer Metastasis Rev. Trauth, B. Science , — McGahon, A. Cotter, T. Cancer Res. Swat, W. Methods , 79— Cell death Download Now Download Download to read offline. Smawi GH Follow. G protein coupled receptor.

G protein signal. Transport Of Proteins. Necrosis vs apaptosis. Cell junctions ppt. Related Books Free with a 30 day trial from Scribd. Elizabeth Howell. Logicomix: An epic search for truth Apostolos Doxiadis.

Related Audiobooks Free with a 30 day trial from Scribd. Pastoral Song James Rebanks. Sanjay Kumar. Adarsh Maurya. Dikaha Gupta. Ridham Patel.

Chukwunyere Nwachhi. Yasmin Ahmad , Student at university of dyiala. Rajesh Srivastava. Show More. Views Total views. Actions Shares. No notes for slide. Cell death 1. Did you know? Sharefa Shaker 3. Cell cycle regulation 6. So there has to exist mechanisms for ensuring other cells in the body are removed, when appropriate. Normal cell Small blebs Big blebs,no organelles Cell membrane ruptures, organelles non functional Apoptotic bodies, organelles functional, no inflammation DNA fragmentati on, organelles in the blebs Small blebs necrosis apoptosis Inflammation of surrounding tissues 8.

Necrosis Causes Necrosis may occur due to external or internal factors: External factors may involve 1. Mechanical trauma physical damage to the body that causes cellular breakdown 2. Damage to blood vessels which may disrupt blood supply to associated tissue , 3.

Thermal effects extremely high or low temperature can result in necrosis due to the disruption of cells. Causes Internal factors causing necrosis include 1.