This work was supported by NIH Grants AG043376 (Project 2 and Core A, PDR; Project 1 and Core B, LJN) and AG056278 (Project 3 and Core A, PDR; and Project 2, LJN) and a grant from the Glenn Foundation (LJN).

Animal use was approved by the Scripps Florida Institutional Animal Care and Use Committee.
1. Generation of senescent murine embryonic fibroblast (MEF) &#8211; 12-15 days
<ol>
	<li>Isolate wild type and Ercc1-/- MEFs from pregnant female mice at embryonic day 13 (E13) as described previously33. 
	NOTE: All following steps are carried out in a tissue culture hood under aseptic conditions and using sterile instruments.</li>
	<li>Resect the embr...

SA-&#946;-Gal is a well-defined biomarker for cellular senescence originally discovered by Dimri&#160;et al. (1995) showing that senescent human fibroblasts have increased activity of SA-&#946;-Gal when assayed at pH 623 compared to proliferating cells. Meanwhile, in vitro and in vivo assay for SA-&#946;-Gal have been established for different cell types and tissues25,39,40. The fluorescence base...

Cellular senescence was described for the first time by Leonard Hayflick and Paul Moorhead, who showed that normal cells had a limited ability to proliferate in culture1. Senescent cells fail to proliferate despite the presence of nutrients, growth factors and lack of contact inhibition, but remain metabolically active2. This phenomenon is known as replicative senescence and was mainly attributed to the telomere shortening, at least in human cells3. Further studies have shown that cells can also be induced to undergo senescence in response to other stimuli, such as oncogenic stress (oncogene induced senescence, OIS), DNA damage, cytotoxic drugs, or irradiation (stress induced senescence, SIS)4,5,6. In response to DNA damage, including telomere erosion, cells either senesce, start uncontrolled cell growth, or undergo apoptosis if the damage cannot be repaired. In this case, cell senescence seems to be beneficial as it acts in a tumor suppressive manner2. In contrast, senescence is increased with aging due to the accumulation of cellular damage including DNA damage. Since senescent cells can secrete cytokines, metalloproteinases and growth factors, termed the senescence-associated secretory phenotype (SASP), this age-dependent increase in cellular senescence and SASP contributes to decreased tissue homeostasis and subsequently aging. Also, this age-dependent increase in the senescence burden is known to induce metabolic diseases, stress sensitivity, progeria syndromes, and impaired healing7,8 and is, in part, responsible for the numerous age-related diseases, such as atherosclerosis, osteoarthritis, muscular degeneration, ulcer formation, and Alzheimer&#39;s disease9,10,11,12,13. Eliminating senescent cells can help to prevent or delay tissue dysfunction and extend healthspan14. This has been shown in transgenic mouse models14,15,16 as well as by using senolytic drugs and drug combinations that were discovered through both drug screening efforts and bioinformatic analysis of pathways induced specifically in senescent cells17,18,19,20,21,22. Identifying more optimal senotherapeutic drugs, able to more effectively reduce the senescent cell burden, is an important next step in the development of therapeutic approaches for healthy aging.
Senescent cells show characteristic phenotypic and molecular features, both in culture and in vivo. These senescence markers could be either the cause or the result of senescence induction or a byproduct of molecular changes in these cells. However, no single marker is found specifically in senescent cells. Currently, senescence-associated &#946;-galactosidase (SA-&#946;-Gal) detection is one of the best-characterized and established single-cell based methods to measure senescence in vitro and in vivo. SA-&#946;-Gal is a lysosomal hydrolase with an optimal enzymatic activity at pH 4. Measuring its activity at pH 6 is possible because senescent cells show increased lysosomal activity23,24. For living cells, increased lysosomal pH is obtained by lysosomal alkalinization with the vacuolar H+-ATPase inhibitor Bafilomycin A1 or the endosomal acidification inhibitor chloroquine25,26. 5-Dodecanoylaminofluorescein Di-&#946;-D-galactopyranoside (C12FDG) is used as substrate in living cells as it retains the cleaved product in the cells due to its 12 carbon lipophilic moiety25. Importantly, SA-&#946;-Gal activity itself is not directly connected with any pathway identified in senescent cells and is not necessary to induce senescence. With this assay, senescent cells can be identified even in the heterogeneous cell populations and aging tissues, such as skin biopsies from older individuals. It also has been used to show a correlation between cell senescence and aging23 as it is a reliable marker for senescent cell detection in several organisms and conditions27,28,29,30. Here, a high throughput SA-&#946;-Gal screening assay based on the fluorescent substrate C12FDG using primary mouse embryonic fibroblasts (MEFs) with robustly oxidative stress induced cell senescence is described and its advantages and disadvantages are discussed. Although this assay can be performed with different cell types, the use of Ercc1-deficient, DNA repair impaired MEFs allows for more rapid induction of senescence under conditions of oxidative stress. In mice, reduced expression of the DNA repair endonuclease ERCC1-XPF causes impaired DNA repair, accelerated accumulation of endogenous DNA damage, elevated ROS, mitochondrial dysfunction, increased senescent cell burden, loss of stem cell function and premature aging, similar to natural aging31,32. Similarly, Ercc1-deficient MEFs undergo senescence more rapidly in culture17. An important feature of the senescent MEF assay is that each well has a mixture of senescent and non-senescent cells, allowing for the clear demonstration of senescent cell-specific effects. However, although we believe that the use of oxidative stress in primary cells to induce senescence is more physiologic, this assay also can be used with cell lines where senescence is induced with DNA damaging agents like etoposide or irradiation.

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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DMEM&#160;</td>
			<td style="width:201px;">Corning</td>
			<td style="width:344px;">10-013-CV</td>
			<td style="width:167px;">medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ham&#39;s F10</td>
			<td>Gibco</td>
			<td>12390-035</td>
			<td style="width:167px;">medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">fetal bovine serum</td>
			<td>Tissue Culture Biologics</td>
			<td>101</td>
			<td style="width:167px;">serum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1x non-essential amino acids</td>
			<td>Corning</td>
			<td>25-025-Cl</td>
			<td style="width:167px;">amino-acids</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">bafilomycin A1&#160;</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td style="width:167px;">lysosomal inhibitor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">C12FDG</td>
			<td>Setareh Biotech</td>
			<td>7188</td>
			<td style="width:167px;">b-Gal substrate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hoechst 33342&#160;</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td style="width:167px;">DNA intercalation agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">17DMAG</td>
			<td>Selleck Chemical LLC</td>
			<td>50843</td>
			<td style="width:167px;">HSP90 inhibitor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">InCell6000 Cell Imaging System</td>
			<td>GE Healthcare</td>
			<td></td>
			<td style="width:167px;">High Content Imaging System</td>
		</tr>
	</tbody>
</table>

sa-&#946;-galactosidase-based screening assay for the identification of senotherapeutic drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&#946;-Gal activity can be detected in cells that are induced to senesce by various ways from replicative exhaustion, genotoxic and oxidative stress, to oncogene activation23,25,38. In the current model using Ercc1-deficient mouse embryonic fibroblast cells, normoxic growth conditions (20% O2) were sufficient to induce cell senescence after cultivating them for a few passag...

Watch this Scientific Journal Video about SA-Î²-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs at JoVE.com

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

We describe here for the first time a method that allows high-throughput drug screening for senotherapeutic compounds that have been shown to be beneficial in aging-related diseases. In fact, several clinical trials with drugs tested in this essence are currently ongoing. The main advantage of this technique is to distinguish between two types of senotherapeutic drugs, senolytics and senomorphics, by using the well-established detection of SA-beta-Galactosidase in senescent cells.This technique is an important screening tool for novel senotherapeutic compounds. In fact, several compounds that have been identified with the help of this screen are featured in various high-profile publications, and are currently in clinical trial. This technique is extremely versatile, and may be adapted to several high-throughput screens.In fact, it's possible to multiplex screens, and screen for compounds that suppress senescence via the activation or inhibition of one of several pathways. Mince each embryo into one millimeter pieces and pipe it up and down several times. Next, add 10 milliliters of growth medium to the tissues of each embryo.Plate them in a 10 centimeter culture dish, and incubate at 37 degrees Celsius in 3%oxygen and 5%carbon dioxide for two to three days. And incubate the rest of each embryo with 0.25%trypsin EDTA mixture for 10 minutes. To trypsinize cells, first, carefully remove the growth medium and wash the cells with 10 milliliters of 1X PBS twice.Then, add two milliliters of a 025 trypsin EDTA solution to the cells, and incubate them at 37 degrees Celsius for two to three minutes. Next, inspect the cells under a microscope to make sure that the cells are detached from the surface. Add the same amount of growth medium to terminate the trypsin digestion, and transfer the cells to a conical tube.Then, centrifuge the cells at 200 times G for three minutes. Discard the supernatant, and add fresh growth medium to re-suspend the cells. Next, count the cells and seed them in new plates at the projected cell density.For non-senescent subcultivation, first, split confluent cells at a one-to-four ratio. Then incubate cells at 37 degrees Celsius in 3%oxygen and 5%carbon dioxide to extend for another passage. Next, to induce cell senescence, seed split confluent cells from passage one at a ratio of one to four and incubate them at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide environment for three days.To monitor cellular senescence, with an advanced Coulter Cell Counter system, measure the gradual increase in cell diameter and cell volume during each trypsinization step. To begin beta-Gal screening assay, seed 5, 000 senescent cells or 3, 000 non-senescent cells per well in 96 well plates. Then, add 100 microliters of growth medium to each well and incubate cells at 37 degrees Celsius and 20%oxygen and 5%carbon dioxide for six hours.Next, add drug dilutions to MEF cells and incubate the cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide for 24 to 48 hours. After incubation, remove the drug solution and wash cells with 100 microliters of 1X PBS. To induce lysosomal alkalinization, pre-treat cells with 90 microliters of bafilomycin A1 solution, diluted in fresh cell culture medium at a final concentration of 100 nanomolar.Then, incubate cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide environment for an hour. For fluorescence analysis of SA-beta-Gal activity, make a fresh working solution of C12FDG diluted in growth medium, at a final concentration of 100 micromolar. Then, add 10 microliters of the working solution to the culture medium at a final concentration of 10 micromolar, and incubate cells at 37 degrees Celsius in 20%oxygen and 5%carbon dioxide for two hours.Wash cells with 100 microliters of 1X PBS. Next, add two microliters of a 100 micrograms-per-milliliter Hoechst 33342 dye at a final concentration of two micrograms per milliliter. Incubate the cells at 37 degrees Celsius, 20%oxygen, and 5%carbon dioxide for 20 minutes.After incubation, remove the media, add 100 microliters of fresh growth medium, and proceed to imaging. In this study, a high-content fluorescent image acquisition and analysis platform allowed for identification of senolytic drugs, such as 17-DMAG, that reduced the overall SA-beta-Gal activities in murine embryonic fibroblast cell cultures containing senescent cells in passage five. The software-generated quantitative analyses of mixtures of senescent and non-senescent cells allow for the clear demonstration of senescent cell-specific effects, and the elimination of background beta-galactosidase activity by bafilomycin A.Make sure all necessary cell controls are included, especially untreated senescent cells and senescent cells treated with a positive control.This essay includes working with primary cells under oxidative stress, which can lead to variation that need to be monitored. This procedure is the first step in the detection of senotherapeutic drugs. Additional cell viability and cell senescent essays need to be performed to confirm the screening results.Several new senolytic drugs have been detected using this essay, including the first senolytic drug combination dasatinib quercetin, heat shock protein 90 inhibitors, Bcl-2 family inhibitors, and the polyphenol fisetin.

SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Abstract