Protocol for quantifying SA-β-gal activity as a measure of senescence in islets of a mouse model of type 1 diabetes

Summary

A subpopulation of pancreatic beta cells becomes senescent during type 1 diabetes (T1D) progression, and removal of these populations protects against T1D in mice. Here, we present a protocol to measure senescence in murine pancreatic islet cells through analysis of senescence-associated β-galactosidase activity. We describe steps for staining with the fluorogenic substrate C₁₂FDG and analysis by flow cytometry. Increased cell size is another marker of senescence and can also be concurrently measured in the same experiment.

For complete details on the use and execution of this protocol, please refer to Lee et al.¹ and Helman et al.²

Graphical abstract

Highlights

• •Protocol to measure senescence in islet cells through SA-β-gal activity
• •Steps for islet cells isolation, dispersion, and flow analysis
• •C₁₂FDG flow-based staining serves as a measurement for SA-β-gal activity

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

A subpopulation of pancreatic beta cells become senescent during type 1 diabetes (T1D) progression, and removal of these populations protects against T1D in mice. Here, we present a protocol to measure senescence in murine pancreatic islet cells through analysis of senescence-associated β-galactosidase activity. We describe steps for staining with the fluorogenic substrate C₁₂FDG and analysis by flow cytometry. Increased cell size is another marker of senescence and can also be concurrently measured in the same experiment.

Before you begin

Senescent beta cells accumulate during the natural progression of T1D in non-obese diabetic (NOD) mice.³ Limiting persistent accumulation of senescent beta cells through immunosurveillance can protect NOD mice against T1D.¹ SA-β-gal, encoded by GLB1, is a lysosomal enzyme that is widely used as a marker of senescence.⁴ SA-β-gal activity is frequently measured by in situ staining using a chromogenic substrate such as X-gal at pH 6.0. Here, we present an alternative protocol that details the staining of dispersed islet cells with a fluorogenic substrate of SA-β-gal (C₁₂FDG) and its analysis via flow cytometry. In principle, this approach can be extended beyond islet cells, yet this will likely require optimization of the conditions.

Institutional permissions

The animal care and experimental procedures were carried out in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee (IACUC). Researchers interested in performing this protocol should acquire prior permissions from their relevant institutions.

Isolation of islets

Timing: 3–6 h

• 1.Isolate pancreatic islets as described previously in Lee et al.⁵

Preparing for islet dispersion

Timing: 5 min

• 2.Set centrifuge (with 5 and 15 mL adapters) temperature to 4°C.
• 3.Set water bath temperature to 37°C.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
TruStain FcX (anti-mouse CD16/32) antibody (1 μg/106 cells)	BioLegend	Cat# 101320; RRID: AB_1574975
APC anti-mouse CD45 antibody (1:100)	BioLegend	Cat# 147708; RRID: AB_2563539
Biological samples
Isolated mouse islets	In-house procedure	For information, see Lee et al., 2020
Chemicals, peptides, and recombinant proteins
Dimethyl sulfoxide	Thermo Fisher Scientific	Cat# BP231-100
C12FDG	Cayman Chemical	Cat# 25583
7-AAD	BioLegend	Cat# 420404
Accutase	Innovative Cell Technologies	Cat# AT-104
Fetal bovine serum	Thermo Fisher Scientific	Cat# F0926
Cell staining buffer	BioLegend	Cat# 420201
Software and algorithms
FlowJo v10	FlowJo, LLC	https://www.flowjo.com
Other
LSRFortessa X-20	BD Biosciences
40 μm cell strainer	Falcon	Cat# 352340
Water bath	VWR	Cat# 76308-896

Materials and equipment

Mouse islet media

Reagent	Final concentration	Amount
RPMI 1640	90%	450 mL
Fetal bovine serum	10%	50 mL
100X antibiotic/antimycotic	1×	5 mL
Total	N/A	505 mL

Store at 4°C for a maximum of 4 weeks.

• •10% FBS in RPMI 1640: add 50 mL of FBS in 450 mL RPMI 1640.

Store at 4°C for a maximum of 1 week.

Step-by-step method details

Islet dispersion

Timing: 1 h

Since pancreatic islets are clusters of cells aggregated together, it is necessary to disperse them into a single cell suspension before performing the flow cytometry experiments. In this section, it is crucial to obtain a high percentage of single cells with high viability.

• 1.Put islets from each mouse into a 60 mm plate with 5 mL of mouse islet media.
• 2.Allow to rest from 1 h to overnight (16 h) in a 5% CO₂ incubator at 37°C.
• 3.
  Move islets from 60 mm plate into a 15 mL tube.

  • a.
    Rinse with 5 mL of PBS to ensure all islets are moved.
• 4.Centrifuge at 200 g for 3 min at room temperature (20°C–24°C) and remove supernatant with aspirator.
• 5.Add 5 mL of PBS to wash the mouse islet media away. Centrifuge at 200 g for 3 min and remove supernatant completely.
• 6.Add 2 mL of Accutase, a cell dissociation solution, in the tube and incubate in a 37°C water bath.
• 7.Set a timer for 30 min. After 15 min, pipet up and down 10x every 5 min, for a total of 4 times.
• 8.Add 10 mL of prewarmed 10% FBS in RPMI 1640 to stop the reaction.
• 9.
  Filter the cell suspension using a 40 μm cell strainer into a 50 mL tube.

  Note: This will get rid of larger aggregates, and potentially separate loosely associated doublets/triplets into single cells.

  • a.
    Rinse filter with additional media to minimize cell loss.
• 10.Spin at 300 g for 5 min at 4°C, and aspirate supernatant.
• 11.
  Wash cells by adding 2 mL of cell staining buffer.

  • a.
    Spin at 300 g for 5 min at 4°C, and aspirate supernatant.
  • b.
    Resuspend pellet in 1 mL cell staining buffer.

Preparation for flow cytometry

Timing: 2 h

C₁₂FDG staining serves as a measurement for SA-β-gal activity. In this section, cells will be stained with APC-CD45 and 7-AAD in addition to C₁₂FDG, allowing for exclusion of dead cells and leukocytes in our flow analysis.

• 12.Add 1.8 μL of C₁₂FDG solution (stock: 16.5 mM) to the tube.
• 13.Incubate the tube in a 37°C water bath for 1 h.
• 14.Spin at 300 g for 5 min at 4°C, and aspirate supernatant.
• 15.
  Add 1 mL of cell staining buffer, and transfer cells to a 5 mL tube.

  • a.
    Rinse tube with 1 mL of additional cell staining buffer to minimize cell loss.
• 16.Spin at 300 g for 5 min at 4°C, and remove supernatant by decanting.

Note: around 100 μL of solution is left after decanting.

• 17.
  Add anti-CD16/32 antibody (1.0 μg per 10⁶ cells) to the tube to block FC receptors.

  • a.
    Incubate for 10 min.
• 18.Add 1 μL of APC anti-CD45 to the tube and incubate on ice for 30 min.

Note: it is not necessary to wash away the anti-CD16/32 antibody after step 17.

• 19.Wash cells by adding 1 mL of cell staining buffer, and spin at 300 g for 5 min at 4°C.
• 20.Decant supernatant and resuspend cells in 250–500 μL of cell staining buffer.
• 21.Add 5 μL of 7-AAD solution to the tube and incubate for 10 min before flow analysis.
• 22.
  Analyze sample with a flow cytometer.

  • a.
    Gating strategy: exclusion of debris, inclusion of single cells, exclusion of dead cells (negative for 7-AAD or other live/dead marker used), and exclusion of leukocytes (CD45-negative).
  • b.
    Lasers used: 7-AAD with 561 nm laser; APC-CD45 with 640 nm laser, C₁₂FDG with 488 nm laser. This set-up may be different depending on the live/dead marker and other fluorophores used.

Expected outcomes

The activity of SA-β-gal is a hallmark of senescence and can be measured by staining cells with C₁₂FDG followed by flow cytometry analysis. C₁₂FDG is a substrate of SA-β-gal and can be cleaved to form a detectable fluorescent product.⁶ During the analysis, after gating for single, viable, and non-leukocyte cells, the comparison of the percentage of positively stained cells from the histogram of C₁₂FDG from each sample can be used to compare the amount of SA-β-gal⁺ cells in each sample (Figure 1). Alternatively, SA-β-gal activity levels between samples can be compared by calculating median fluorescence intensity (MFI) from the C₁₂FDG⁺ gated population. With the same gating strategy, cell size can also be compared by creating a histogram for forward scatter, with cells undergoing senescence having a greater cell size (Figure 2).

Figure 1.

Measurement of SA-β-gal activity in islet cells through C₁₂FDG analysis, related to step 22

(A) Gating strategy for analysis of single and viable cells that are negative for CD45. (B) Representative histogram of C₁₂FDG, where sample 1 exhibits increased senescence compared to sample 2. Fold change calculation for samples is based on median fluorescence intensity (RFI). Data are represented as means ± SEM. ∗∗p<0.01. Unpaired two tail t-test was applied. Figure adapted from Lee et al.¹

Figure 2.

Comparison of islet cell size between samples, related to step 22

Representative histogram of forward scatter, where sample 1 has a subpopulation of cells that are larger. Fold change calculation for samples is based on median forward scatter. Data are represented as means ± SEM. ∗p<0.05. Unpaired two tailed t-test was applied. Figure adapted from Lee et al.¹

Limitations

This protocol aims to quantify SA-β-gal activity as a phenotype of late-stage senescence in non-immune islet cells during T1D progression in the NOD mouse model. However, islet cells are heterogeneous and consist of multiple cell types such as alpha, beta, and delta cells. Therefore, the results of the analysis would also consist of cells other than beta cells. In addition, there are non-endocrine cells in the islet that could be stained, such as endothelial cells. It should also be noted that this protocol will not distinguish between senescent islet cells that arise during aging and those that result from a stress response as SA-β-gal activity can be high in both.

Troubleshooting

Problem 1

Poor viability during flow analysis, related to step 22.

Potential solution

• •Determine if the viability issues are stemming from the dispersion step or the staining step. This can be done with trypan blue staining and cell counting after the dispersion step, or through live/dead dye with flow cytometry.
• •Be wary of being too harsh during islet dispersion with Accutase. If cell viability is low after dispersion, use less force during pipetting or reduce incubation time. Alternatively, a non-enzymatic PBS or HBSS based dissociation buffer can be used (e.g., Thermo Fisher Cat #13150016).
• •Make sure cells are on ice when they are not incubating at 37°C, and ensure appropriate media is prewarmed before adding to cells.

Problem 2

Lack of C₁₂FDG⁺ stained population, related to step 22.

Potential solution

• •C₁₂FDG was stored improperly: Ensure stocks are made of 16.5 mM in DMSO and stored at -20°C and not subject to repeated freeze-thaw cycles.
• •C₁₂FDG concentration was too low: Ensure concentration is ∼33 μM in final solution with cells.

Problem 3

All cells stain C₁₂FDG⁺, related to step 22.

Potential solution

• •C₁₂FDG concentration was too high: Ensure concentration is ∼33 μM in final solution with cells.
• •Incubation with C₁₂FDG too long: Ensure cells are incubated for 1 h at 37°C in a water bath.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Feyza Engin (fengin@wisc.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Hugo Lee (hslee5@wisc.edu).

Materials availability

This protocol did not generate new unique reagents.

Data and code availability

This study did not generate new datasets or code.