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

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

Example protocol

AT A GLANCE

Protocol Summary
  1. Treat samples as desired

  2. Prepare and add Xite™ Red beta-D-galactopyranoside working solution to samples

  3. Incubate samples at 37 °C for 15 to 45 minutes

  4. Monitor the fluorescence intensity with fluorescence microscope using Cy3/TRITC filter set or flow cytometer using 575/26 nm filter (PE channel)

Important

To ensure accurate results, allow all kit components to reach room temperature before beginning 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™ Red beta-D-galactopyranoside Stock Solution (100X)

Add 100 µL of DMSO (Component C) into Xite™ Red beta-D-galactopyranoside (Component A) and mix thoroughly.

Note: Divide any remaining Xite™ Red beta-D-galactopyranoside stock solution into individual single-use aliquots. Store these aliquots at -20°C, protected from light.

PREPARATION OF WORKING SOLUTION

Xite™ Red beta-D-galactopyranoside Working Solution

To prepare the Xite™ Red beta-D-galactopyranoside working solution, mix 10 µL of the Xite™ Red beta-D-galactopyranoside stock solution with 1 mL of Assay Buffer.

Note: Xite™ Red beta-D-galactopyranoside working solution should be used promptly.   

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat your samples as desired.

    Note: For 96 well plate format, grow cells in 100 µL of cell culture medium and treat as desired. Volumes can be adjusted based on the plate size.

  2. Remove the cell culture medium and wash the cells with a 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. The optimum concentration of Bafilomycin A1 may vary on the type of cells.

  3. Add 100 µL of Xite™ Red beta-D-galactopyranoside working solution, and incubate the samples at 37 °C incubator for 15 to 45 minutes.

    Note: The optimal time for incubation needs to be determined carefully.

  4. Remove the working solution and wash cells with buffer of your choice.

    Note: If performing flow cytometry for attached cells, cells can be trypsined at this step and collected in a tube.

  5. Resuspend the cells in the Assay Buffer (Component B) and monitor the fluorescence intensity with a flow cytometer using a 575/26 nm filter (PE channel) or fluorescence microscope using a Cy3/TRITC filter set.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Cell Meter™ Cellular Senescence Activity Assay Kit *Green Fluorescence*4985178000010.79001, 0.9520.320.35

Citations

View all 4 citations: Citation Explorer
Extended replicative lifespan of primary resting T cells by CRISPR/dCas9-based epigenetic modifiers and transcriptional activators
Authors: Huang, Siping and Lau, Cia-Hin and Tin, Chung and Lam, Raymond HW
Journal: Cellular and Molecular Life Sciences (2024): 1--18
TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells
Authors: Scaramuzza, Shaun and Jones, Rebecca M and Sadurni, Martina Muste and Reynolds-Winczura, Alicja and Poovathumkadavil, Divyasree and Farrell, Abigail and Natsume, Toyoaki and Rojas, Patricia and Cuesta, Cyntia Fernandez and Kanemaki, Masato T and others,
Journal: Nature Communications (2023): 5071
Stromal-induced epithelial-mesenchymal transition induces targetable drug resistance in acute lymphoblastic leukemia
Authors: Park, Chun Shik and Yoshihara, Hiroki and Gao, Qingsong and Qu, Chunxu and Iacobucci, Ilaria and Ghate, Pankaj S and Connelly, Jon P and Pruett-Miller, Shondra M and Wagner, Ben and Robinson, Camenzind G and others,
Journal: Cell Reports (2023): 112804
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

References

View all 50 references: Citation Explorer
ES5 is involved in the regulation of phosphatidylserine synthesis and impacts on early senescence in rice (Oryza sativa L.).
Authors: Rani, Mohammad Hasanuzzaman and Liu, Qunen and Yu, Ning and Zhang, Yingxin and Wang, Beifang and Cao, Yongrun and Zhang, Yue and Islam, Md Anowerul and Zegeye, Workie Anley and Cao, Liyong and Cheng, Shihua
Journal: Plant molecular biology (2020): 501-515
Exercise enhances skeletal muscle regeneration by promoting senescence in fibro-adipogenic progenitors.
Authors: Saito, Yuki and Chikenji, Takako S and Matsumura, Takashi and Nakano, Masako and Fujimiya, Mineko
Journal: Nature communications (2020): 889
Ginsenoside Rg1 Inhibits Cell Proliferation and Induces Markers of Cell Senescence in CD34+CD38- Leukemia Stem Cells Derived from KG1α Acute Myeloid Leukemia Cells by Activating the Sirtuin 1 (SIRT1)/Tuberous Sclerosis Complex 2 (TSC2) Signaling Pathway.
Authors: Tang, Yan-Long and Zhang, Cheng-Gui and Liu, Heng and Zhou, Yue and Wang, Ya-Ping and Li, Yuan and Han, Yan-Jun and Wang, Cui-Li
Journal: Medical science monitor : international medical journal of experimental and clinical research (2020): e918207
LINC00623/miR-101/HRAS axis modulates IL-1β-mediated ECM degradation, apoptosis and senescence of osteoarthritis chondrocytes.
Authors: Lü, Guohua and Li, Lei and Wang, Bing and Kuang, Lei
Journal: Aging (2020)
Autolysosomal degradation of cytosolic chromatin fragments antagonizes oxidative stress-induced senescence.
Authors: Han, Xiaojuan and Chen, Honghan and Gong, Hui and Tang, Xiaoqiang and Huang, Ning and Xu, Weitong and Tai, Haoran and Zhang, Gongchang and Zhao, Tingting and Gong, Chuhui and Wang, Shuang and Yang, Yu and Xiao, Hengyi
Journal: The Journal of biological chemistry (2020)
Page updated on November 24, 2024

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Catalog Number23007
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Spectral properties

Excitation (nm)

544

Emission (nm)

567

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform

Flow cytometer

Excitation488 nm laser
Emission575, 26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom

Components

Fixability test with Cell Meter™ Cellular Senescence Activity Assay Kit using a NovoCyte Flow Cytometer (ACEA Biosciences). 9L-LacZ cells were incubated with DMSO or Xite™ Red beta-D-galactopyranoside for 45 mins at 37 °C. The signal before and after fixation was acquired using PE channel. (Cells were then fixed with 4% formaldehyde for 20 minutes at room temperature, and wash once.) 
Fixability test with Cell Meter™ Cellular Senescence Activity Assay Kit using a NovoCyte Flow Cytometer (ACEA Biosciences). 9L-LacZ cells were incubated with DMSO or Xite™ Red beta-D-galactopyranoside for 45 mins at 37 °C. The signal before and after fixation was acquired using PE channel. (Cells were then fixed with 4% formaldehyde for 20 minutes at room temperature, and wash once.) 
Fixability test with Cell Meter™ Cellular Senescence Activity Assay Kit using a NovoCyte Flow Cytometer (ACEA Biosciences). 9L-LacZ cells were incubated with DMSO or Xite™ Red beta-D-galactopyranoside for 45 mins at 37 °C. The signal before and after fixation was acquired using PE channel. (Cells were then fixed with 4% formaldehyde for 20 minutes at room temperature, and wash once.) 
Fluorescence images with 9L-LacZ cells. 9L-LacZ cells were stained with Xite™ Red beta-D-galactopyranoside directly in cell culture medium 45 mins at 37 °C. The signal was acquired using Cy3/TRITC filter set.