Cell Meter™ Cellular Senescence Activity Assay Kit Green Fluorescence

Cellular senescence was measured with Cell Meter™ Cellular Senescence Activity Assay Kit using a NovoCyte Flow Cytometer (ACEA Biosciences). HL-60 cells were incubated with Camptothecin for 6 hours to induce senescence and stained with Xite™ beta-D-galactopyranoside for 30 mins at 37<sup>o</sup>C. The signal was acquired using FITC channel in ACEA NovoCyte flow cytometer.

Cellular senescence was measured with Cell Meter™ Cellular Senescence Activity Assay Kit using a fluorescence microscope. HeLa cells were incubated with Camptothecin for 6 hours to induce senescence and stained with Xite™ beta-D-galactopyranoside for 30 mins at 37C. The signal was acquired using FITC filter set.

9L-LacZ cells  were stained with DMSO or FDG or Xite™ beta-D-galactopyranoside for 30 minutes at 37C incubator, an then the cells were washed twice and scrapped using a scrapper with 1 mL HH buffer. Cells were collected and analyzed with flow cytometer using FITC channel.

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Spectral properties

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Storage, safety and handling

Intended use

Research Use Only (RUO)

Alternative formats

Cell Meter™ Cellular Senescence Activity Assay Kit *Red Fluorescence*

Overview SDS Protocol

Absorbance (nm)

487

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.35

Extinction coefficient (cm ^-1 M ^-1)

80000¹

Excitation (nm)

498

Emission (nm)

517

Quantum yield

0.7900¹, 0.95²

Cellular Senescence is an irreversible growth arrest triggered in order to prevent growth in DNA damaged cells. Senescence-associated beta-galactosidase (SA-beta-gal) is highly overexpressed in senescent cells and it has been widely used as a senescence marker. X-gal staining, a colorimetric method is widely available and used to detect SA-beta-gal in senescent cells. The color method has some limitations such as requirement of fixation of samples due to the low cell permeability of X-gal, longer staining time and low sensitivity. Cell Meter™ Cellular Senescence Activity Assay Kit uses Xite™ beta-D-galactopyranoside, a fluorogenic beta-Gal substrate that readily enters into live cells, and gets cleaved by SA-β-gal inside cells, generating strong green fluorescence. Unlike cell-impermeable X-Gal substrate, it has excellent cell permeability. Cell Meter™ Cellular Senescence Activity Assay Kit enables users to detect the senescence with higher sensitivity with robust performance. The Xite product is well retained inside the cells, producing a stable signal for fluorescence imaging and flow cytometry analysis.

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

Excitation	FITC filter set
Emission	FITC filter set
Recommended plate	Black wall/clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Treat samples as desired
Prepare and add Xite™ beta-D-galactopyranoside working solution to samples
Incubate samples at 37 °C for 15 to 45 minutes
Monitor the fluorescence intensity using flow cytometer with 530/30 nm filter (FITC channel)

Important Thaw each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Xite™ beta-D-galactopyranoside stock solution (100X)

Add 100 uL DMSO (Component C) into Xite™ beta-D-galactopyranoside (Component A) and mix well. Note: Store the unused Xite™ beta-D-galactopyranoside stock solution at -20 °C in single use aliquots.

PREPARATION OF WORKING SOLUTION

Xite™ beta-D-galactopyranoside working solution (1X)

Dilute 10 uL of Xite™ beta-D-galactopyranoside stock solution (100X) with 1 mL of Assay Buffer to make Xite™ beta-D-galactopyranoside working solution (1X). Note: Xite™ beta-D-galactopyranoside working solution should be used promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Protocol

Treat your samples as desired.
Wash the cells with buffer of your choice such as DPBS. Note: For selectively tracking β-Gal in live cells, cells can be treated with Bafilomycin A1 for blocking endogenous β-Gal. Optimum concentration of Bafilomycin A1 may vary on type of cells.
Add 100 uL Xite™ beta-D-galactopyranoside working solution for 15-45 minutes and incubate the samples at 37°C incubator. Note: Optimal time for incubation needs to be determined carefully.
Remove the working solution and wash cells with buffer of your choice.
Resuspend the cells in the Assay Buffer (Component B) and monitor the fluorescence intensity with flow cytometer using 530/30 nm filter (FITC channel) or fluorescence microscope with FITC filter set.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Absorbance (nm)	487
Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	80000¹
Excitation (nm)	498
Emission (nm)	517
Quantum yield	0.7900¹, 0.95²

Product Family

Name	Excitation (nm)	Emission (nm)
Cell Meter™ Cellular Senescence Activity Assay Kit Red Fluorescence	544	567

Images

Figure 1. Cellular senescence was measured with Cell Meter™ Cellular Senescence Activity Assay Kit using a NovoCyte Flow Cytometer (ACEA Biosciences). HL-60 cells were incubated with Camptothecin for 6 hours to induce senescence and stained with Xite™ beta-D-galactopyranoside for 30 mins at 37^oC. The signal was acquired using FITC channel in ACEA NovoCyte flow cytometer.

Figure 2. Cellular senescence was measured with Cell Meter™ Cellular Senescence Activity Assay Kit using a fluorescence microscope. HeLa cells were incubated with Camptothecin for 6 hours to induce senescence and stained with Xite™ beta-D-galactopyranoside for 30 mins at 37C. The signal was acquired using FITC filter set.

Figure 3. 9L-LacZ cells were stained with DMSO or FDG or Xite™ beta-D-galactopyranoside for 30 minutes at 37C incubator, an then the cells were washed twice and scrapped using a scrapper with 1 mL HH buffer. Cells were collected and analyzed with flow cytometer using FITC channel.

Citations

View all 46 citations: Citation Explorer

Merkel Cell Polyomavirus Large T Antigen Induces Cellular Senescence for Host Growth Arrest and Viral Genome Persistence through Its Unique Domain
Authors: Pham, Alexander M and Ortiz, Luz E and Lukacher, Aron E and Kwun, Hyun Jin
Journal: Cells (2023): 380

Topological DNA damage, telomere attrition and T cell senescence during chronic viral infections
Authors: Ji, Y., Dang, X., Nguyen, L. N. T., Nguyen, L. N., Zhao, J., Cao, D., Khanal, S., Schank, M., Wu, X. Y., Morrison, Z. D., Zou, Y., El Gazzar, M., Ning, S., Wang, L., Moorman, J. P., Yao, Z. Q.
Journal: Immun Ageing (2019): 12

Cell senescence, apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis
Authors: Lorda-Diez, C. I., Solis-Mancilla, M. E., Sanchez-Fern and ez, C., Garcia-Porrero, J. A., Hurle, J. M., Montero, J. A.
Journal: J Anat (2019): 815-829

Dynamic transcriptome profiling in DNA damage-induced cellular senescence and transient cell-cycle arrest
Authors: Zhao, Z., Dong, Q., Liu, X., Wei, L., Liu, L., Li, Y., Wang, X.
Journal: Genomics (2019): ersion="1.0" encoding="UTF-8" ?>23005.enlEndN

Stochastic modeling of aging cells reveals how damage accumulation, repair, and cell-division asymmetry affect clonal senescence and population fitness
Authors: Song, R., Acar, M.
Journal: BMC Bioinformatics (2019): 391

Regulatory T cells trigger effector T cell DNA damage and senescence caused by metabolic competition
Authors: Liu, X., Mo, W., Ye, J., Li, L., Zhang, Y., Hsueh, E. C., Hoft, D. F., Peng, G.
Journal: Nat Commun (2018): 249

The Novel Small Molecule STK899704 Promotes Senescence of the Human A549 NSCLC Cells by Inducing DNA Damage Responses and Cell Cycle Arrest
Authors: Park, C. W., Bak, Y., Kim, M. J., Srinivasrao, G., Hwang, J., Sung, N. K., Kim, B. Y., Yu, J. H., Hong, J. T., Yoon, D. Y.
Journal: Front Pharmacol (2018): 163

Leucine reduces the proliferation of MC3T3-E1 cells through DNA damage and cell senescence
Authors: da Luz Dias, R., Basso, B., Donadio, M. V. F., Pujol, F. V., Bartrons, R., Haute, G. V., Gassen, R. B., Bregolin, H. D., Krause, G., Viau, C., Saffi, J., Nunes, F. B., Rosa, J. L., de Oliveira, J. R.
Journal: Toxicol In Vitro (2018): 1-10

Hydrogen Treatment Protects against Cell Death and Senescence Induced by Oxidative Damage
Authors: Han, A. L., Park, S. H., Park, M. S.
Journal: J Microbiol Biotechnol (2017): 365-371

MicroRNA-16 feedback loop with p53 and Wip1 can regulate cell fate determination between apoptosis and senescence in DNA damage response
Authors: Issler, M. V. C., Mombach, J. C. M.
Journal: PLoS One (2017): e0185794