at 4 C. This working solution is stable for few hours
2. Trypsinization of cells before the assay should be moderated because exaggerated trypsinization can induce additional
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_14, © Springer Science+Business Media New York 2017
Chapter 14
Detection of Senescent Cells by Extracellular Markers Using a Flow Cytometry-Based Approach
Mohammad Althubiti and Salvador Macip
Abstract
Senescence is a cellular process that is thought to have prognostic and therapeutic relevance in conditions such as cancer, aging, and fi brosis. However, current protocols for identifying senescent cells in vitro and in vivo have several drawbacks. Most markers used lack suffi cient specifi city and false positives and nega- tives in common. In addition, classical staining techniques often require lengthy protocols and do not offer objective quantifi cation. Recently, several novel markers of senescence associated with the plasma mem- brane have been identifi ed. Here, we propose to take advantage of these markers to defi ne a customizable FACS-based protocol to detect senescent cells using antibodies tagged with fl uorescence dyes. This method has the advantage of being fast and allowing quantitation. Furthermore, its specifi city is increased using several markers simultaneously.
Key words Senescence , Extracellular markers , Antibodies , Flow cytometry , SA-β-Gal
1 Introduction
Cell senescence is an irreversible cell cycle arrest that acts as a tumor suppressor mechanism [ 1 ]. Accumulation of senescent cells in tissues can be seen in response to DNA damage , oncogene up- regulation, or other kinds of stress, as well as in normal aging [ 2 ]. It is accepted that senescence plays a central role in the functional changes associated with old age and, because of this, it has been considered one of the nine hallmarks of ageing [ 3 ]. This has been confi rmed in a mouse model in which preventing senescent cell accumulation prolongs lifespan [ 4 ]. It has also been shown that senescent cells are up-regu- lated in several pathologies, such as fi brosis, diabetes, obesity, COPD, and other chronic diseases [ 5 , 6 ]. Although their exact role in these conditions is not always clear, they are believed to be involved in the pathogenesis in many of them [ 7 – 10 ].
The clinical relevance of senescence has enhanced the interest in studying these cells. However, no absolute markers of senescence
have yet been defi ned, although many nonspecifi c have been described [ 11 ]. Currently, the accepted standard method of iden- tifi cation of senescent cells is staining for the expression of Senescence-associated β-galactosidase (SA-β-gal) [ 12 ]. This enzyme has the particularity of being active at low pH and is highly expressed in senescent cells, although it is not known to have any relationship to the mechanisms involved in triggering or maintain- ing the phenotype. The SA-β-gal protocol, described for the fi rst time 20 years ago, has been shown to have many false positive and negative results, and may require up to 15 h to complete [ 12 – 14 ].
Also, its scoring depends on the visual identifi cation of stained cells, which facilitates human error and bias. Thus, there is a need for a more streamlined method with increased specifi city, reduced processing time, and objective quantitation.
We present a novel system of senescent cell identifi cation using a straightforward fl uorescence-activated cell sorting-based approach. It is based on the expression on the cell surface of previously characterized markers of senescent cells [ 15 – 18 ].
Two or more commercially available antibodies , tagged with fl uorescence dyes, are used simultaneously to recognize their extracellular domains. This does not require permeabilization of the cells, which accelerates the protocol, and the quantitative readout is proportional to the fl uorescence intensity detected in a sample. By adding markers from a selected list, the protocol can be adjusted for the tissue of interest and its specifi city can be increased.
2 Materials
1. 96 Rounded bottom multiwell plates ( see Note 1 ).