SenezRedTM Labeling Agent

Distinguish senescent MSC cells from healthy ones

$150.00$700.00
  • Quantity : 20-100 assays
  • Shelf life : 1 Year
  • Store at : -20 oC

SenezRedTM Labeling Agent

$150.00$700.00

Distinguish senescent MSC cells from healthy ones

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Overview

SenezRedTM can be used for identifying, staining and imaging senescent MSC cells from different sources. SenezRedTM is a membrane-permeable fluorescent probe which selectively stains live, senescent mesenchymal stem cells. SenezRedTM -labeled cells can be visualized using fluorescent imaging. Efficacy demonstrated on umbilical cord MSCs, bone marrow MSCs and adipose MSCs.

SenezRedTM is the only labeling agent available in the market for live staining of senescent MSCs.

Applications include:

  1. Simple and rapid labeling protocol.
  2. Enables selective labeling of primary senescent, less-potent mesenchymal stem cells without fixation.
  3. Can be used to label live cells for fluorescent imaging.
  4. Can be potentially used as a process analytical tool for stem cell bioprocesses, and to track MSC in migration assays, co-culture systems, cell-cell communications and tumor tropism studies.
  5. As part of a standardized QC protocol.

This product is for research use only and is not for use in diagnostic procedures.

Protocols

Use a red microscope filter to view SenezRedTM stained cells. Adjust the contrast so that the brighter cells are distinct against the lighter red background. Initially perform the beta-galactosidase assay as a benchmark.

Refer to video.


Additional Equipment/Reagents Required

  1. hMSCs growth media (e.g. α-MEM + 10% FBS + antibiotics)
  2. Hoechst stain
  3. Fluorescence microscope with Cy5 filter (e.g. barrier filter 663 nm to 738 nm, excitation filter 590 nm – 650 nm)

General Protocol

  1. Aseptically aliquot appropriate amount of SenezRed™ to reach 1µM final concentration in 1ml of hMSC growth media, in a 15-mL tube. 1mL of hMSC growth media containing SenezRed™ is needed per well of a 6-WP
  2. hMSCs seeded in 6-well plates can be stained with SenezRed™ when they reach 50% - 60% confluency
  3. Aseptically remove spent hMSC growth media from 6-well plates by pipetting
  4. Add 1mL of hMSC growth media containing SenezRed™ to each well of a 6-WP
  5. Incubate the cells at 37oC, 5% CO2 for 1 hour
  6. Aseptically remove hMSC growth media containing SenezRed™ from 6-well plates by pipetting
  7. Add 1mL of fresh hMSC growth media + Hoechst to each well of a 6-WP
  8. Incubate the cells at 37oC, 5% CO2 for 1 hour
  9. SenezRed™ stained cells can be images using a fluorescent microscope using a Cy5 filter (Ex/Em: 631/673 nm) (Fig.1)

SenezRed Comparison

Figure 1 shows fluorescence microscopy image of proliferative and senescent hMSC stained with SenezRed™.

NOTE:

  • It is recommended to optimize SenezRed™ concentration for each cell type under study.
  • It is critical to maintain equal exposure time for samples and controls for optimal staining results.
  • Senescent cells can be generated by long-term in vitro culture >10 passages or more and validated with SenezRed™ and senescence associated β-galactosidase staining.

Validating SenezRed™ Staining and Associated β-Galactosidase Staining

  1. The underside of 6-WP with SenezRed™ stained MSC can be marked into four quadrants (demo)
  2. SenezRed™ stained cells can be imaged using a fluorescent microscope using a Cy5 filter (Ex/Em: 631/673 nm) and imaged areas can be circled.
  3. After imaging, remove spent media from SenezRed™ stained cells
  4. Follow manufacturer’s recommendation for performing senescence associated β-gal staining
  5. Marked areas of 6-WP that were imaged for SenezRed™ can be imaged under bright-field to observe presence of senescence associated β-gal staining (Fig. 2)

SenezRed co-staining

Figure 2 shows microscopic images of hMSC co-stained with SenezRed™ and β-gal staining.

NOTE:

  • Fluorescence microscope with associated imaging software may also be used to record co-ordinates of SenezRed™ stained cells. These co-ordinates can be re-used for bright-field imaging, for comparison with senescence associated β-gal staining.

General Protocols for Staining SenezRed™ in Microcarrier Culture

  1. Aseptically aliquot appropriate amount of SenezRed™ to reach 1µM final concentration in 1ml of hMSC growth media, in a 15-mL tube. 1mL of hMSC growth media containing SenezRed™ is needed per well of a 6-WP
  2. Aseptically aliquot 1mL of samples from MSC-microcarrier culture into one well of a 6-WP
  3. Aseptically remove spent hMSC growth media from 6-well plates by pipetting
  4. Add 1mL of hMSC growth media containing SenezRed™ to each well of a 6-WP
  5. Incubate the cells at 37oC, 5% CO2 for 1 hour
  6. Aseptically remove hMSC growth media containing SenezRed™ from 6-well plates by pipetting
  7. Add 1mL of fresh hMSC growth media + Hoechst to each well of a 6-WP
  8. Incubate the cells at 37oC, 5% CO2 for 1 hour
  9. SenezRed™ stained cells can be images using a fluorescent microscope using a Cy5 filter (Ex/Em: 631/673 nm) (Fig.3)

Senezred label

Figure 3 shows fluorescence microscopy image of microcarrier cultures of proliferative and senescent hMSC stained with SenezRed™.

NOTE:

  • It is recommended to optimize SenezRed™ concentration for each cell type under study.
  • It is critical to maintain equal exposure time for samples and controls for optimal staining results.

Download Detailed Protocol

Videos

Protocol

Live Stain

Publications

1. Oh, Steve et al. “Brilliant vital fluorescent probes for detecting stem cell proliferation and senescence”. Stem Cell Society Singapore (SCSS) Symposium 2015, Singapore. 17-19 Nov 2015.


2. Raghothaman, Deepak et al. “A cell permeable fluorescent probe for detecting stem cell senescence”. Biology of Aging conference, Singapore. 22-24 October, 2015.


3. Raghothaman, Deepak et al. “Vital fluorescent probes for detecting live stem cell proliferation and senescence”. Aging: Cellular Mechanisms and Therapeutic Opportunities, a Herrenhausen Symposium. A Nature Medicine Conference. Hanover, Germany. 28-29 September, 2015.


4. Oh, Steve. “Stem cell bioprocesses and senescence”. The Rejuvenation Biotechnology Conference, San Francisco, USA. 18-21 Aug. 2015.