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

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.
Ordering information
Price ()
Catalog Number23007
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
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
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OverviewpdfSDSpdfProtocol


Excitation (nm)
544
Emission (nm)
567
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.

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


Component A: Xite™ Red beta-D-galactopyranoside1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (100 µL)

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

PREPARATION OF WORKING SOLUTION

Xite™ Red beta-D-galactopyranoside working solution
Dilute 10 µL of Xite™ Red beta-D-galactopyranoside stock solution with 1 mL of Assay Buffer to make Xite™ Red beta-D-galactopyranoside working solution. Note: Xite™ Red beta-D-galactopyranoside working solution should be used promptly.   

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat your samples as desired.
  2. 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.
  3. Add 100 µL Xite™ Red 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.
  4. Remove the working solution and wash cells with buffer of your choice.
  5. Resuspend the cells in the Assay Buffer (Component B) and monitor the fluorescence intensity with flow cytometer using 575/26 nm filter (PE channel) or fluorescence microscope using Cy3/TRITC filter set. 

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)544
Emission (nm)567

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, 0.9520.320.275

References


View all 50 references: Citation Explorer
Chronic Mild Stress Modified Epigenetic Mechanisms Leading to Accelerated Senescence and Impaired Cognitive Performance in Mice.
Authors: Puigoriol-Illamola, Dolors and Martínez-Damas, Mirna and Griñán-Ferré, Christian and Pallàs, Mercè
Journal: International journal of molecular sciences (2020)
miR‑20b inhibits the senescence of human umbilical vein endothelial cells through regulating the Wnt/β‑catenin pathway via the TXNIP/NLRP3 axis.
Authors: Dong, Feifei and Dong, Shaohua and Liang, Ying and Wang, Ke and Qin, Yongwen and Zhao, Xianxian
Journal: International journal of molecular medicine (2020): 847-857
The Arabidopsis Senescence Associated Gene 13 Regulates Dark-Induced Senescence and Plays Contrasting Roles in the Defense Against Bacterial and Fungal Pathogens.
Authors: Dhar, Nikhilesh and Caruana, Julie and Erdem, Irmak and Subbarao, Krishna V and Klosterman, Steven J and Raina, Ramesh
Journal: Molecular plant-microbe interactions : MPMI (2020)
Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.
Authors: Panda, Prashanta Kumar and Patra, Srimanta and Naik, Prajna Paramita and Praharaj, Prakash Priyadarshi and Mukhopadhyay, Subhadip and Meher, Biswa Ranjan and Gupta, Piyush Kumar and Verma, Rama S and Maiti, Tapas K and Bhutia, Sujit K
Journal: Journal of cellular physiology (2020): 2776-2791
KLF2 induces the senescence of pancreatic cancer cells by cooperating with FOXO4 to upregulate p21.
Authors: Yuedi, Dai and Houbao, Liu and Pinxiang, Lu and Hui, Wang and Min, Tao and Dexiang, Zhang
Journal: Experimental cell research (2020): 111784
The potential role of senescence in limiting fibrosis caused by aging.
Authors: Meng, Xinghua and Wang, Haoran and Song, Xiaopeng and Clifton, Alancia C and Xiao, Jianhua
Journal: Journal of cellular physiology (2020): 4046-4059
Cell senescence and fibrotic lung diseases.
Authors: Liu, Rui-Ming and Liu, Gang
Journal: Experimental gerontology (2020): 110836
The novel protein CSAP accelerates leaf senescence and is negatively regulated by SAUL1 in the dark.
Authors: So, Won Mi and Kim, Soo Youn and Hyoung, Sujin and Shin, Jeong Sheop
Journal: Plant cell reports (2020): 325-334
The Importance of Stem Cell Senescence in Regenerative Medicine.
Authors: Khademi-Shirvan, Maliheh and Ghorbaninejad, Mahsa and Hosseini, Samaneh and Baghaban Eslaminejad, Mohamadreza
Journal: Advances in experimental medicine and biology (2020)
The role of ascorbic acid in rice leaf senescence and photo-carbon imbalance.
Authors: Yu, Le and Zhang, Qilei and Lu, Lina and Gao, Hui and Liu, Qiang and Liu, Yonghai and Yang, Chengwei and Peng, Changlian
Journal: Functional plant biology : FPB (2020)