Cellular Senescence Detection, Reimagined

Quantitative, Rapid and Reliable SA-β-gal Analysis for Modern Research

Cellular senescence is a cornerstone of ageing, oncology, and immunology research. Yet, traditional detection methods remain time-consuming, subjective, and poorly suited for quantitative workflows. The SPiDER-βGal technology addresses these limitations by enabling highly sensitive, quantitative, and versatile detection of senescence-associated β-galactosidase (SA-β-gal) in both live and fixed cells. Whether your objective is high-throughput screening, single-cell analysis, or functional validation, this solution adapts to your workflow, not the other way around.

Why SA-β-gal Detection Matters

Senescence-associated β-galactosidase (SA-β-gal) is a widely accepted biomarker due to its overexpression in senescent cells. However, endogenous β-galactosidase activity in living cells often compromises specificity.

How We Solve This: 

• Bafilomycin A1 inhibits lysosomal ATPase
• Maintains neutral lysosomal pH
• Eliminates interference from endogenous β-galactosidase
• Ensures selective and accurate SA-β-gal detection in live cells

Figure 1: Cellular Senescence Detection Kit – SPiDER-βGal allows to detect SA-β-gal with high sensitivity and ease of use. SPiDER-βGal is a new reagent to detect β-galactosidase which possesses a high cell-permeability and a high retentivity inside cells. SA-β-gal are detected specifically in living cells by using a reagent (Bafilomycin A1) to inhibit endogenous β-galactosidase activity. Therefore, SPiDER-βGal can be applied to quantitative analysis by flow cytometry.

Figure 2: Cellular Senescence Detection Kit ,SPiDER-βGal experimental set up.

Two Kits, One Seamless Workflow

SG03: Cellular Senescence Detection Kit (Immunofluorescence)

Designed for microscopy and flow cytometry applications

SG05: Cellular Senescence Plate Assay Kit

Optimised for plate reader-based quantification and high-throughput screening

Key Benefits for Your Research

Fluorescence Characteristics

• Quantitative results instead of subjective visual counting
• Rapid staining (~30 minutes) to accelerate workflows
• High cell permeability and retention for stronger, stable signals
• Multi-platform compatibility:
  • Microscopy
  • Flow cytometry
  • Plate reader
• Flexible use with both live and fixed cells

• Excitation: 500–540 nm
• Emission: 530–570 nm
• Bright, stable fluorescence signal for reliable quantification

Figure 3: Ex/Em Spectra - Ex: 500-540 nm and Em: 530-570 nm

Performance Compared to Conventional Methods

Application Data

1. Co-staining in WI-38 Cells

• Comparison between early (P1) and senescent (P10) cells
• Multiplex staining:
  • SA-β-gal (senescence)
  • γ-H2AX (DNA damage)
  • DAPI (nuclei)
• Clear increase in senescence and DNA damage markers with passage number

Figure 4: WI-38 cells at passage 1 VS passage 10. a)  SA-β-gal produced by senescent cells are labeled, b) DNA Damage staining with γ-H2AX, c) Nuclear localisation with DAPI and d) merged image.

Figure 5: Staining Conditions.

Figure 6: Comparison between SG03 (left) and X-Gal method (right). Using SG03 method, researchers were able to quantify senescent cells in Menin KO T-Cells using flow cytometry.

2. Senescence Detection in T Cells

• Detection of induced senescence in CD8+ T cells
• Enables quantification via flow cytometry
• Demonstrates superiority over X-Gal:
  • Quantitative
  • Faster
  • Compatible with suspension cells

3. Lysosomal Activity & pH in Senescence

• Senescence induced using doxorubicin (DOX)
• Simultaneous analysis of:
  • SA-β-gal activity
  • Lysosomal mass
  • Lysosomal pH
• Consistent results across microscopy and plate reader quantification

Figure 7: An increase in lysosomal mass and pH acidification was observed in senescence-induced cells, and the normalised fluorescence intensity of lysosomal mass and pH by plate reader measurement showed the same result.

Designed Around Your Needs

Go Further

You need:

• Reliable quantification → We deliver reproducible fluorescence readouts
• Faster workflows → Results in under 30 minutes
• Flexible platforms → One assay, multiple detection systems
• Scalability → From single experiments to high-throughput screening

From basic discovery to drug screening, the SPiDER-βGal platform provides the precision, speed, and flexibility required in modern cellular senescence research.

Move beyond qualitative staining. Quantify senescence with confidence.

Discover our comprehensive guide to cellular senescence research products and explore all the solutions available for your experiments.

For a quotation or further information, please do not hesitate to contact us.

Technical FAQ

• Are there any advices when observing the senescent cells?

  Lipofuscin is a fluorescent pigment that accumulates in a variety of cell types with age. Lipofuscin consists of autofluorescent granules and may results in high background for fluorescence microscopy. In order to achieve accurate SA-β-gal activity assay in senescent cells, we recommend to prepare samples without SPiDER-βGal staining. Please compare fluorescence intensity of both cells with or without SPiDER-βGal staining.

  For Flow Cytometry Detection

  Step 1. Prepare senescent cells and non-senescent cells. Measure MFI (Mean Fluorescence Intensity) of samples below.

  Senescent cells
  Sample A: The cells stained with SPiDER-βGal
  Sample B: The cells without SPiDER-βGal staining

  Non-senescent cells
  Sample A’: The cells stained with SPiDER-βGal
  Sample B’: The cells without SPiDER-βGal staining

  Step 2. Calculate SA-β-gal activity (senescent cells) with the following formula
  SA-β-gal activity (senescent cells) = MFI of Sample A - MFI of Sample B

  Step 3. Calculate SA-β-gal activity (non-senescent cells) with the following formula:
  SA-β-gal activity (non-senescent cells) = MFI of Sample A’ - MFI of Sample B’

  Determine the SA-β-gal activity by comparing the SA-β-gal activity between senescent cells and non-senescent cells.
  Change of SA-β-gal activity associated with senescence = (Value from Step 2- value from Step 3)

  For Microscopy

  Step 1. Prepare senescent cells without SPiDER-βGal staining and observe fluorescent image.
  Step 2. Adjust detection sensitivity in microscopy to reduce background autofluorescence of lipofuscin.
  Step 3. Observe fluorescent image of senescent cells and non-senescent cells under the settled condition in step 2.

• What if I see background in control cells?

  Check if reagents were stored correctly. SPiDER-βGal DMSO stock solution and Bafilomycin A1 DMSO stock solution are stable for 1 month at -20 ℃. SPiDER-βGal working solution and Bafilomycin A1 working solution can’t be stored. Be sure to use the working solution immediately.

• What if I see background in senescent cells without adding SPiDER-βGal working solution?

  Lipofuscin, which consists of autofluorescent granules, accumulated in the senescent cells. In order to achieve accurate SA-β-gal activity assay in senescent cells, we recommend preparing samples without SPiDER-βGal staining. Please compare fluorescence intensity of both cells with / without SPiDER-βGal staining.

• What if I see background in fixed cells assay?

  The incubation with SPiDER-βGal working solution was done in a 5% CO2 incubator. We recommend not to use a 5% CO2 incubator during incubation with SPiDER-βGal working solution. If incubation is done in a 5% CO2 incubator, the pH of the buffer may become acidic. Acidic pH results in higher background from the endogenous β-galactosidase activity and it would be difficult to distinguish between control cells and senescent cells. Please incubate the plate in a dry incubator without CO2.

• No difference in fluorescence intensity between senescent cells and control cells

  Cellular senescence wasn’t induced. Please make sure to prepare a positive control.

• Low fluorescence Reading

  • Incubation time with SPiDER-βGal working solution was short. Optimize the incubation time (～ 60 min).
  • Concentration of SPiDER-βGal working solution was low. Test different ranges.

• Background in fix cells assay

  We recommend not to use a 5% CO2 incubator during incubation with SPiDER-βGal working solution. If incubation is done in a 5% CO2 incubator, the pH of the buffer may become acidic. Acidic pH results in higher background from the endogenous β-galactosidase activity and it would be difficult to distinguish between control cells and senescent cells. Please incubate the plate in a dry incubator without CO2.

• Reference

  
  Article content created by Tebubio using courtesy materials provided by Dojindo Europe.