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Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965,DOI 10.1007/978-1-62703-239-1_9, © Springer Science+Business Media, LLC 2013

Chapter 9

Chemiluminescent Detection of Senescence-Associated b Galactosidase

Vinicius Bassaneze , Ayumi Aurea Miyakawa , and José Eduardo Krieger

Abstract

Identifying molecules that serve as markers for cell aging is a goal that has been pursued by several groups. Senescence-associated β galactosidase (SA- β gal) staining is broadly used and very easily detected. β -gal is a lysosomal enzyme strongly correlated to the progression of cell senescence. Here, we describe a simple, fast, and quantitative protocol to quantify SA- β gal activity in cell lysate extracts by a chemiluminescent method using galacton as substrate.

Key words: Cell senescence , Chemiluminescence , Quantitative method , SA- β gal

Despite the fact that senescence-associated β galactosidase (SA- β gal) is not a causative element to cell senescence ( 1 ) , its accumulation is irreversibly augmented in senescent cells and it is reliable and easy to detect. Moreover, there is no truly speci fi c marker of senescence nor does each senescent cell express all possible senescence markers ( 2 ) . Generally, SA- β gal activity is quanti fi ed by the X-gal staining procedure, a semiquantitative and subjective procedure that demands a laborious counting process of cells stained in variable intensities, even in a homogeneous cell culture. The distinction between faint and negative staining is often dif fi cult to observe, making the methodology inaccurate. To overcome these limita-tions, new methodologies to detect and quantify β -gal activity using speci fi c substrates and claiming to be more quantitative and less time consuming have been proposed (Table 1 ).

The methodology described in this chapter uses galacton ® (Fig. 1 ), a 1,2-dioxetane substrate, that is non-isotopic and it is known to provide high signal intensity, low background, and high

1. Introduction

158 V. Bassaneze et al.

sensitivity, and it is compatible with multiple assay formats (tubes, microwell plates, or X-ray fi lms) when used to detect prokaryotic β -galactosidase. It was originally designed for the detection of prokaryotic β -gal enzyme (pH 7.8) used as a reporter in transfec-tion studies ( 3 ) . The methodology was adapted to detect and quantify SA- β gal activity in mammalian cell culture by modifying the pH 7.8 to pH 6.0. In fact, chemiluminescent substrates usually provide a more sensitive result than fl uorescent or colorimetric probes, particularly when the amount of samples is limited ( 4 ) . Therefore, we present methods in detail for the detection and quanti fi cation of SA- β gal using the galacton ® chemiluminescent substrate.

● Tissue culture dishes and fl asks and cell scrappers. Speci fi c plastic tubes for Monolight 2010 chemiluminometer ●

(Analytical Luninescence, San Diego, CA, USA).

● Chemiluminometer (Monolight 2010, Analytical Luminescence). Spectrophotometer (Victor Wallac 1420, Perking Elmer, ●

Wellesley, MA, USA).

2. Materials

2.1. Disposables

2.2. Equipment

Table 1 Different methods for SA- b gal detection

Substrate type Application References

Chromogenic X-gal Adherent cell cultures and tissue samples

( 5, 6 )

Fluorescent C 12 FDG Cell culture supernatant or fl ow cytometry

( 7– 10 )

MUG Cell culture extracts ( 11 )

Chemiluminescent Galacton ® Cell culture extracts ( 12 )

Fig. 1. Galacton ® structure. (3-(4-methoxyspiro(1,2-dioxetane-3,20-(50-chloro)-tricy-clo-(3.3.1.13,7)decan)-4-yl)phenyl-b- D -galactopyranoside)) structural formula.

1599 SA-bgal Detection in Cell Lysates

● Human saphenous vein smooth muscle cells ( see Note 1 ). Dulbecco’s modi fi ed Eagle’s medium (DMEM) with 4.5 g/L ●

glucose. Fetal bovine serum (FBS). ●

Penicillin and streptomycin, 100×. ●

Phosphate buffer saline (PBS): 0.8% w/v sodium chloride, ●

0.02% KCl, 1.14% sodium phosphate dibasic heptahydrate, and 0.024% potassium phosphate dibasic (pH 7.4). 0.25% trypsin/1 mM EDTA (w/v). ●

Magnesium chloride salt. ●

Potassium ferricyanide. ●

Citric acid. ●

Monobasic sodium phosphate salts. ●

Dibasic sodium phosphate salts. ●

Gelatin from porcine skin (Sigma-Aldrich, St. Louis, MO, ●

USA). X-gal (Invitrogen, Eugene, CA, USA). ●

100× Galacton ● ® chemiluminescent substrate (3-(4-metho-xyspiro(1,2-dioxetane-3,20-(50-chloro)-tricyclo-(3.3.1.13,7)decan)-4-yl)phenyl-b- D -galactopyranoside) (Applied Biosystems, Carlsbad, CA, USA). 10× Emerald Luminescence Enhancer for Solution Assays ●

(Applied Biosystems). Cell culture lysis buffer (Promega, Madison, WI, USA). ●

Bradford protein determination (Bio-Rad, Hercules, CA, ●

USA).

● Prepare 6-well plates coated with gelatin. Therefore add a thin layer of 1% gelatin from porcine skin in each well. Incubate the plates (without cover) at room temperature inside a laminar fl ow and let water completely evaporate. Plates can be wrapped with plastic fi lm, stored in the refrigera- ●

tor, and used on the next day. Inside a laminar fl ow, rinse the veins with PBS in order to ●

remove the remaining blood. Open it longitudinally and cut it in small pieces (1–4 mm 2 ) using sterile scissors, on a plastic petri dish. Place six pieces (explants), with the lumen face down, inside of ●

each well and a small drop (approximately 50 μ L) of cell culture

2.3. Reagents

3. Methods

3.1. Preparation of Smooth Muscle Cell Cultures

160 V. Bassaneze et al.

medium supplemented with 1% penicillin/streptomycin and 20% FBS on each explant and maintain the culture in 5% CO 2 atmosphere at 37°C for at least 24 h. The following day the explants should have attached to the ●

support; therefore carefully add 1 mL of cell culture medium to (the border of) each well. Cell culture medium should be replaced every 2–3 days and ●

the proliferations should be observed under a light microscope. It usually takes at least 2 weeks to observe a considerable cell proliferation, which is the moment when the explant must be removed. After 25–30 days cell cultures should have reached con fl uence. ●

At this moment the cells can be frozen for later usage, or replated and expanded, for experimentation or comparisons with earlier passages.

● Discard the cell culture medium and wash the cells twice with PBS, 1 mL per well. Aspirate PBS, add 500 ● μ L per well of 0.25% trypsin/1 mM EDTA, and incubate for at most 3 min at the incubator. Check under microscope if the cells detached. ●

Add 1.5 mL per well of cell culture medium and transfer all ●

content to a 15 mL plastic tube. Count the number of cells and note the period between pas- ●

sages for further population doubling accountability. Transfer 8 × 10 ● 4 cells per well and maintain in a 37°C incubator with 5% CO 2 . For expansion, transfer from 0.8 × 10 ● 4 to 1.2 × 10 4 cells/cm 2 , depending on cell yield and fl ask size.

● Discard the cell culture medium and wash the cells twice with PBS, 1 mL per well. Aspirate PBS completely, add 500 ● μ L per well of 0.25% trypsin/1 mM EDTA, and incubate for at most 3 min at the incubator, checking under microscope if the cells detached. Add 1.5 mL per well of cell culture medium and transfer all ●

content to a 15 mL plastic tube. Count the number of cells and spin the cells at 500 × ● g for 3 min. Discard the supernatant and resuspend the cells with cell cul- ●

ture medium to a concentration of 1 × 10 6 cells/mL.

3.2. Passaging Smooth Muscle Cells

3.3. Stock Preparation

1619 SA-bgal Detection in Cell Lysates

Transfer 900 ● μ L of cell culture medium to a cryovial and add 100 μ L of DMSO. Mix gently. Put the vials in −80°C freezer for no more than 1 month. For ●

long-term storage, cells should be transferred to liquid nitro-gen containers.

● Perform all experiment in duplicate at room temperature. Two days after platting the cells, lyse the cells using 300 ● μ L cell culture lysis buffer. Use a cell scraper to facilitate detaching and transfer the content to 1.5 mL tubes ( see Note 2 ). Prepare the reaction solution by diluting galacton ● ® substrate 1:100 in 100 mM sodium phosphate buffer (pH 6.0), supple-mented with 20 μ M magnesium chloride. The reaction solution must be prepared upon use. Prepare enough for immediate use. Incubate 5 ● μ L of cell culture lysate in 200 μ L reaction solution inside a plastic tube suitable for analysis by means of a lumi-nometer. Incubate at room temperature for 45 min. Prepare the Emerald Luminescence Ampli fi er Solution by add- ●

ing 600 μ L of 5 N NaOH and 300 μ L of 10× Emerald Enhancer to a fi nal volume of 2.1 mL milli-Q water. Prepare enough for immediate use. After 45 min of reaction, add 300 ● μ L of Emerald Luminescence Ampli fi er Solution to the tube and immediately perform the reading on luminometer, for 10 s (Fig. 2 ). The result should be normalized by total protein content ( see Note 3 ).

3.4. SA- b gal Activity Determination

Fig. 2. Reaction steps.

162 V. Bassaneze et al.

● Perform a 1:10 dilution of the cell lysate with milli-Q water. Pipet 10 μ L of diluted sample in a 96-well plate. It is recom-mended to do this in triplicate. Create a standard curve using bovine serum albumin (BSA) ●

ranging from 0.05 to 0.4 μ g/ μ L. Add 10 μ L of each on a 96-well plate, also in triplicate. Prepare the diluted Bradford dye reagent (1:5) and add 200 ● μ L of the diluted to each well if possible using a multichannel pipet. Incubate at room temperature for at least 5 min. ●

Measure absorbance at 595 nm on a spectrophotometer at no ●

more than 1 h. Multiply the concentration obtained by 5 ● μ L (volume used to react the samples on SA- β gal activity determination). Use the corresponding values to normalize the luminescence measurement, obtaining fi nal data as a.u./ μ g protein ( see Notes 4 and 5 ).

1. Experiments involving human materials must be carried out conforming to institutional regulations for biosafety and ethics.

2. Material can be stored at −80°C for future assay. It is strongly recommended to perform the SA- β gal activity of all samples you want to compare in the same assay.

3. This protocol is adjusted for smooth muscle cell. The assay may need to be standardized for each cell type. The basal SA- β gal varies between cell lines.

4. As mentioned before, the basal SA- β gal varies between cell lines. For that reason, the comparison of SA- β gal content between different cell types is not suitable for determining which one is more senescent.

5. To avoid confounding factors, different experiments should be performed with the same con fl uence (preferably not fully con fl uent) and without serum deprivation.

References

3.5. Total Protein Content Determination

4. Notes

1. Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (2006) Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 5:187–195

2. Campisi J (2011) Cellular senescence: putting the paradoxes in perspective. Curr Opin Genet Dev 21:107–112

3. Bronstein I, Martin CS, Fortin JJ, Olesen CE, Voyta JC (1996) Chemiluminescence: sensitive detection technology for reporter gene assays. Clin Chem 42:1542–1546

4. Jain VK, Magrath IT (1991) A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation

1639 SA-bgal Detection in Cell Lysates

of beta-galactosidase in lacZ-transfected cells. Anal Biochem 199:119–124

5. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A biomarker that identi fi es senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367

6. Shlush LI, Itzkovitz S, Cohen A, Rutenberg A, Berkovitz R, Yehezkel S, Shahar H, Selig S, Skorecki K (2011) Quantitative digital in situ senescence-associated beta-galactosidase assay. BMC Cell Biol 12:16

7. Kurz DJ, Decary S, Hong Y, Erusalimsky JD (2000) Senescence-associated (beta)-galactosi-dase re fl ects an increase in lysosomal mass dur-ing replicative ageing of human endothelial cells. J Cell Sci 113(Pt 20):3613–3622

8. Noppe G, Dekker P, de Koning-Treurniet C, Blom J, van Heemst D, Dirks RW, Tanke HJ, Westendorp RG, Maier AB (2009) Rapid fl ow cytometric method for measuring senescence

associated beta-galactosidase activity in human fi broblasts. Cytometry A 75:910–916

9. Yang NC, Hu ML (2004) A fl uorimetric method using fl uorescein di-beta- D -galactopy-ranoside for quantifying the senescence-associ-ated beta-galactosidase activity in human foreskin fi broblast Hs68 cells. Anal Biochem 325:337–343

10. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senes-cence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4:1798–1806

11. Gary RK, Kindell SM (2005) Quantitative assay of senescence-associated beta-galactosi-dase activity in mammalian cell extracts. Anal Biochem 343:329–334

12. Bassaneze V, Miyakawa AA, Krieger JE (2008) A quantitative chemiluminescent method for studying replicative and stress-induced prema-ture senescence in cell cultures. Anal Biochem 372:198–203