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Comment
. 2024 Mar 15;5(1):102923.
doi: 10.1016/j.xpro.2024.102923. Epub 2024 Feb 29.

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

Hugo Lee  1 , Peter J Thompson  2 , Feyza Engin  3
Affiliations
Comment

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

Hugo Lee et al. STAR Protoc. .

Abstract

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.

Keywords: Cell Biology; Cell culture; Cell isolation; Metabolism; Model Organisms.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
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.
Figure 2
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.
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References

    1. Lee H., Sahin G.S., Chen C.W., Sonthalia S., Cañas S.M., Oktay H.Z., Duckworth A.T., Brawerman G., Thompson P.J., Hatzoglou M., et al. Stress-induced beta cell early senescence confers protection against type 1 diabetes. Cell Metab. 2023;35:2200–2215.e9. doi: 10.1016/j.cmet.2023.10.014. - DOI - PMC - PubMed
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    1. Lee B.Y., Han J.A., Im J.S., Morrone A., Johung K., Goodwin E.C., Kleijer W.J., DiMaio D., Hwang E.S. Senescence-associated β-galactosidase is lysosomal β-galactosidase. Aging Cell. 2006;5:187–195. doi: 10.1111/j.1474-9726.2006.00199.x. - DOI - PubMed
    1. Lee H., Engin F. Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing. STAR Protoc. 2020;1 doi: 10.1016/j.xpro.2020.100144. - DOI - PMC - PubMed

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