Flow cytometry validated antibodies against receptors of the extrinsic apoptosis pathway. Long incubation times may alter the apoptosis levels and intracellular staining requires fixation and permeabilization of cells. Staining protocols may also need to be optimized. Dead cells can bind antibodies non-specifically so a viability dye is a must to avoid false positives. Note that care should be taken when immunophenotyping apoptotic cells. Table 1 below lists Bio-Rad’s death receptor antibodies validated in flow cytometry. To help with identification, cells were counterstained with CD45 ( MCA87A647). The level of CD95 /FAS receptor ( MCA1539FITC) was determined in B, lymphocytes, C, monocytes and D, granulocytes. A, lymphocytes, monocytes and granulocytes in the live cell fraction were identified by FSC and SSC. This enables you to obtain information on the levels of induction of apoptosis in specific cells.įig.1. CD95 expression in peripheral blood populations. Alternatively specific lineage markers can be used like CD3 for T cells, CD19 for B cells and CD14 for monocytes in an immunophenotyping panel. CD95/FAS (Figure 1) can be determined in various cell types simply by FSC (forward) and SSC (side scatter)profiles where lymphocytes, monocytes and granulocytes can be identified. Flow cytometry is ideal to determine expression levels of these receptors in cell populations from various samples. Understanding the balance between death receptor expression patterns and anti-apoptotic mechanisms, signaling pathways and upregulation of these receptors is important in the fields of cancer, inflammation, tissue healing, transplantation and cell death.
Our Apoptosis Overview page goes into more detail about the two main apoptosis pathways. While it is not possible to measure intrinsically mediated apoptosis induction by flow cytometry, measuring extrinsically mediated apoptosis induction is relatively straightforward as it involves the analysis of the so-called death receptors (DR) CD95, CD261 (DR4), CD262 (DR5), CD120a, and CD120b, which can be detected using antibodies.
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To help you plan and optimize your flow cytometry experiments download or request a copy of our Flow Cytometry Basics Guide. We will also give you tips to avoid common pitfalls and help you choose the right assay. Here we will walk you through specific flow cytometry assays with examples that will enable you to detect apoptosis.
Careful experimental set-up and interpretation of results will allow you to make the most of your experiment.įlow cytometry allows the study of all aspects of apoptosis from induction via surface receptors, to late stages where DNA fragmentation occurs. Other applications such as western blotting or imaging may need to be used. However, flow cytrometry may not be the most appropriate application. The powerful technique of flow cytometry can be used to both detect and quantify the level of apoptosis in a population of cells at static points or in a time course. For instance, if most of your cells are entering into late apoptosis you may not be able to detect early stages of apoptosis with some reagents. Before starting your experiments it is important to note that apoptosis is not a static process and certain tests are time specific. It offers the ability to study large numbers cells individually rather than a mixed population, examining simultaneously the expression of many proteins like cell specific markers and indicators of apoptosis. Flow cytometry is one of the most popular and versatile applications for studying apoptosis.
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