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Imaging examples using GlycoYELLOW™-βGal

  • For live-cell imaging and for fixed cells
  • High sensitivity
  • Membrane permeability and cell retention

Imaging radiation-induced senescence by GlycoYELLOW™-βGal

Screening of β-galactosidase activity by GlycoYELLOW™-βGal

Imaging radiation-induced senescence by GlycoYellow™-βGal

Data were provided by Keiji SUZUKI, Ph.D, Department of Radiation Medical Sciences Atomic Bomb Disease Institute, Nagasaki University.


Live cell imaging of radiation-induced senescent cells using GlycoYELLOW™-βGal
Yellow signal indicates β-galactosidase (β-Gal) activity in living cells.

 

Live cell imaging movie showing β-Gal activity in thyroidal gland follicular cells.
You can see red color of β-Gal in the cytoplasm, after 15 seconds.
Nuclei are stained by Fucci, a cell cycle indicator. Nuclei in red indicate that the cell cycle is arrested in the G1 phase. Cell-cycle arrest was induced by 10 Gy irradiation.

 

Staining of living thyroidal gland follicular cells

Radiation-induced β-Gal (yellow) activity in living thyroidal gland follicular cells can be detected by GlycoYELLOW™-βGal.
In this condition, almost 100% cells are positive in X-gal staining.

 

In contrast, cells not stained by GlycoYELLOW™-βGal survive and grow (bottom left region). This result indicates that GlycoYELLOW™-βGal can detect radiation-induced senescence (SA-β-Gal).

 

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Screening of β-galactosidase activity by GlycoYELLOW™-βGal

Summary

β-galactosidase (β-Gal) activity has been widely used as a senescent marker (SA-βGal) or reporter gene such as lacZ. Conventional X-Gal staining, analysis of β-Gal activity at single-cell level is difficult due to poor sensitivity.  GlycoYELLOW™-βGal, a highly sensitive fluorescent probe for β-Gal activity enables single-cell level detection with high S/N ratio. This probe is applicable for both live and fixed cells. Therefore, it is suitable for pharmacological tests, including cell imaging-based screening system as well as detection of cellular senescence. To test the possibility, we analyzed β-Gal activity of LacZ-transfected cells at single-cell level using Operetta™ (PerkinElmer , USA).

Cell staining protocol

Fixation and staining of HEK293 cells (either LacZ-gene transfected,ie LacZ+, or not ie  LacZ-)

 Fixation and staining of cell
  1. Coat 96 well plate with 50 µL poly-L-lysine and incubate plate at 37℃ overnight in a 5% CO2 incubator.
  2. Remove poly-L-lysine and wash 96 well plate by HBSS twice.
  3. Dispense HEK293 cells to 96 well plate (5×104 cell/ml, 200 µl/well) and incubate for 37℃ overnight in a 5% CO2 incubator.
    ※   Recommendation: Remove cell aggregates using cell strainer (40 µm).

  4. Dissolve GlycoYELLOW™-βGal 1 vial (50 µg) in 93.3 µL DMSO to prepare 1 mM stock solution.
  5. Dilute 1 mM stock solution with HBSS to 5 µM (Cell staining solution).
  6. Add 5000 times diluted Hoechst33342 (1 mg/ml) for nuclear staining to the cell staining solution.
  7. Remove the culture medium on the dish and wash cells gently once by HBSS.
  8. Add 200 µl of the cell staining solution to the dish and incubate at 37°C for 60 min.
  9. Remove the culture medium on the dish and gently wash the cells twice with the observation buffer or with the culture medium.

  10. Fix stained cells for 10 to 30 minutes with 3% of paraformaldehyde.
  11. Fixation for more than 30 minutes is not recommended. Stronger fixation conditions may weaken the fluorescent signal.
  12. After the fixing, gently wash the cells once by HBSS.
  13. Add new HBSS to start observation. Operetta™ (PerkinElmer) was used for the observation.

※ Cells were harvested on 96 Costar flat bottom plate, pictured with 10x long WD.
※ Nuclei (Hoechst33342): ex. 360-400 nm/em. 410-480 nm
  GlycoYELLOW™-βGal: ex. 520-550 nm/em. 560-630 nm

 Imaging analysis of GlycoYELLOW™-βGal stained cell

Figure 1. Microscopy images of HEK LacZ (-) and HEK LacZ (+) cell

Figure 1. Microscopy images of HEK LacZ (-) and HEK LacZ (+) cell

Blue: Nuclei (Hoechst33342)
Yellow: SA-βGal (GlycoYELLOW™-βGal)

The fluorescence intensity of GlycoYELLOW™-βGal was much higher in the LacZ expressing cells (HEK LacZ (+)) than that in the non-expressing cells (HEK LacZ (-)).

 

Figure 2. The result of imaging analysis via Harmony software on Operetta™

Figure 2. The result of imaging analysis via Harmony software on Operetta™

 

(Left) Comparing averaged fluorescence intensity of LacZ(+/-) cells.
HEK LacZ (+) cells showed more than 7.5 times higher fluorescence intensity compare to that of HEK LacZ (-) cells.
(Right) The number of analyzed cells

Number of cells are obtained by counting the number of nuclei. The number of cells are sufficient for the statistical analysis.

Cell imaging and analysis

The Images were acquired using Operetta™ High Contents Imaging System (PerkinElmer, Hopkinton, MA) with 10x objective with appropriate optical filters. The obtained images were analyzed with Harmony™ software (PerkinElmer). In brief, the individual cells in each field of view (FOV) were recognized by using prebuild software functions as follows: DAPI stained nuclei were recognized by “Find Nuclei” function, and then cytoplasmic region were subsequently detected by “Find Cytoplasm” function. The fluorescence intensity derived from GlycoYELLOWTM-βGal in cytoplasm region was quantitated in every cell in FOV. Each step of the image analysis is summarized in Figure 3.

 

Figure 3. The analysis mechanism of Harmony™

Figure 3. The analysis mechanism of Harmony™

 

Input image: Representing image of GlycoYELLOW™-βGal stained cells acquired on Operetta™.
Find Nuclei: Nuclei recognized in input image with “Find Nuclei” function.
Find Cytoplasm: Cytoplasm regions recognized in input image with “Find Cytoplasm” function.
Quantitation: GlycoYELLOW™-βGal positive signal detected in the FOV.

 

Contact Information

Further information or questions on our products, please contact us
E-mail: info_itnl@goryochemical.com