. 2024 Mar 15;5(1):102929.

doi: 10.1016/j.xpro.2024.102929. Epub 2024 Mar 8.

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Sophia Magkouta¹, Dimitris Veroutis², Athanasios Pousias³, Angelos Papaspyropoulos², Kety Giannetti⁴, Natassa Pippa⁵, Nikolaos Lougiakis³, Konstantinos Kambas⁶, Nefeli Lagopati⁷, Aikaterini Polyzou⁸, Maria Georgiou³, Maria Chountoulesi⁹, Stergios Pispas¹⁰, Spyros Foutadakis¹¹, Efthymios Kyrodimos¹², Nicole Pouli³, Panagiotis Marakos³, Athanassios Kotsinas⁸, Panayotis Verginis¹³, Dimitrios Valakos¹¹, Giannis Vatsellas¹⁴, Russell Petty¹⁵, Dimitris Thanos¹⁶, Marco Demaria¹⁷, Konstantinos Evangelou⁸, Raffaella Di Micco⁴, Vassilis G Gorgoulis¹⁸

Affiliations

¹ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Marianthi Simou and G.P. Livanos Labs, 1st Department of Critical Care and Pulmonary Services, School of Medicine, National & Kapodistrian University of Athens, ''Evangelismos'' Hospital, 10676 Athens, Greece.
² Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
³ Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, 15771 Athens, Greece.
⁴ San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
⁵ Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece; Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 11635 Athens, Greece.
⁶ Hellenic Pasteur Institute, 11521 Athens, Greece.
⁷ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Laboratory of Biology, Department of Basic Medical Sciences, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece.
⁸ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece.
⁹ Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece.
¹⁰ Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 11635 Athens, Greece.
¹¹ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹² First ENT Department, Hippocration Hospital, National Kapodistrian University of Athens, 11527 Athens, GR, Greece.
¹³ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Laboratory of Immune Regulation and Tolerance, Division of Basic Sciences, University of Crete Medical School, 70013 Heraklion, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 70013 Heraklion, Greece.
¹⁴ Greek Genome Center, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹⁵ Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
¹⁶ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Greek Genome Center, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹⁷ European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen, Groningen 9713 AV, The Netherlands.
¹⁸ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK; Faculty Institute for Cancer Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M20 4GJ, UK; Faculty of Health and Medical Sciences, University of Surrey, Surrey GU2 7YH, UK. Electronic address: vgorg@med.uoa.gr.

PMID: 38460134
PMCID: PMC10943059
DOI: 10.1016/j.xpro.2024.102929

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Sophia Magkouta et al. STAR Protoc. 2024.

. 2024 Mar 15;5(1):102929.

doi: 10.1016/j.xpro.2024.102929. Epub 2024 Mar 8.

Authors

Affiliations

¹ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Marianthi Simou and G.P. Livanos Labs, 1st Department of Critical Care and Pulmonary Services, School of Medicine, National & Kapodistrian University of Athens, ''Evangelismos'' Hospital, 10676 Athens, Greece.
² Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
³ Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, 15771 Athens, Greece.
⁴ San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
⁵ Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece; Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 11635 Athens, Greece.
⁶ Hellenic Pasteur Institute, 11521 Athens, Greece.
⁷ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Laboratory of Biology, Department of Basic Medical Sciences, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece.
⁸ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece.
⁹ Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece.
¹⁰ Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 11635 Athens, Greece.
¹¹ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹² First ENT Department, Hippocration Hospital, National Kapodistrian University of Athens, 11527 Athens, GR, Greece.
¹³ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Laboratory of Immune Regulation and Tolerance, Division of Basic Sciences, University of Crete Medical School, 70013 Heraklion, Greece; Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 70013 Heraklion, Greece.
¹⁴ Greek Genome Center, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹⁵ Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
¹⁶ Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Greek Genome Center, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece.
¹⁷ European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen, Groningen 9713 AV, The Netherlands.
¹⁸ Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece; Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK; Faculty Institute for Cancer Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M20 4GJ, UK; Faculty of Health and Medical Sciences, University of Surrey, Surrey GU2 7YH, UK. Electronic address: vgorg@med.uoa.gr.

PMID: 38460134
PMCID: PMC10943059
DOI: 10.1016/j.xpro.2024.102929

Abstract

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.¹.

Keywords: Cancer; Cell Biology; Cell isolation; Cell-based Assays; Flow Cytometry; Molecular/Chemical Probes.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests Pending patents: (1) Greek Patent Application 20230100019 and (2) University of Dundee 20230100019, regarding SBB analogs hydrophilic compounds on chemical synthesis, method(s), and application(s) use.

Figures

**Figure 1**
GLF16 staining of lung tissue sections and cellular models of induced senescence (A) Representative pictures of GLF16 staining of lungs of bleomycin (right) or vehicle (left) treated mice. Objectives 10× and 40×, Scale bars: 50 μm (-Bleomycin), 10 μm (+Bleomycin). Pictures were originally published in Magkouta, Veroutis, Pousias, Papaspyropoulos et al., 2023). (Β) Representative pictures of Li-Fraumeni^p21WAF1/Cip1 Tet-ON cells treated with doxycycline (right) or not (left). Lipofuscin aggregates are clearly visualized by GLF16, as depicted by white arrows. Objectives 40×, Scale bar: 10 μm.

**Figure 2**
Flow cytometry application of GLF16 Representative gating strategy to identify senescent cells based on GLF16 staining profile. Total cell populations may be first gated on a forward scatter (FSC)/side scatter (SSC) plot so that debris is excluded from the analysis. Singlets should be subsequently identified using an FSC-A/FSC-H scatter plot. GLF16-positive and -negative populations can be subsequently selected either by GLF16 alone or combined with other markers, such as the proliferation marker Ki67.

**Figure 3**
Representative pictures of expected outcomes upon (A) GLF16 and p16^INK4A or Ki67 fluorescent staining of fixed cells (upper panel) or tissues known to be abundant in senescent cells [lower panel (pictures were originally published in Magkouta, Veroutis, Pousias, Papaspyropoulos et al., 2023)] Objectives (i, ii) 20×, 40×, (iii) 63×, (iv, v) 40×. Scale bars (i-iii) 10 μm, (iv, v) 25 μm). (B) Comparative flow cytometry analysis of senescent THP-1 cells (treated with 200 nM Cytarabine every other day for 7 days) and respective controls (untreated cells) using (i) GLF16 or (ii) C12FDG compounds. (C) Representative flow cytometry analysis of cells incubated with m-GLF16. (D) *In vivo* (left) and *ex vivo* (right) monitoring of senescence in the bleomycin-induced lung fibrosis model, Lu: Lung, Li: Liver, S: Spleen, H: Heart, K: Kidney.

See this image and copyright information in PMC

References

1. Magkouta S., Veroutis D., Pousias A., Papaspyropoulos A., Pippa N., Lougiakis N., Kambas K., Lagopati N., Polyzou A., Georgiou M., et al. A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells. Mol. Cell. 2023;83:3558–3573.e7. doi: 10.1016/j.molcel.2023.09.006. - DOI - PubMed
1. Sabath N., Levy-Adam F., Younis A., Rozales K., Meller A., Hadar S., Soueid-Baumgarten S., Shalgi R. Cellular proteostasis decline in human senescence. Proc. Natl. Acad. Sci. USA. 2020;117:31902–31913. doi: 10.1073/pnas.2018138117. - DOI - PMC - PubMed
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1. Papaspyropoulos A., Hazapis O., Altulea A., Polyzou A., Verginis P., Evangelou K., Fousteri M., Papantonis A., Demaria M., Gorgoulis V. Decoding of translation-regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence. Aging Cell. 2023;22 doi: 10.1111/acel.13893. - DOI - PMC - PubMed
1. Gorgoulis V., Adams P.D., Alimonti A., Bennett D.C., Bischof O., Bishop C., Campisi J., Collado M., Evangelou K., Ferbeyre G., et al. Cellular Senescence: Defining a Path Forward. Cell. 2019;179:813–827. doi: 10.1016/j.cell.2019.10.005. - DOI - PubMed

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One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Affiliations

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources