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Cited by 196Telomeres, Telomerase, and SenescenceGenetics, Aging, and Longevity in Model OrganismsSingle-cell and spatial transcriptomics

Algorithmic assessment of cellular senescence in experimental and clinical specimens

Open access
Kohli, J., Wang, B., Brandenburg, S. M., Basisty, N., Evangelou, K., Varela-Eirin, M., Campisi, J., Schilling, B. +2 more·2021·Nature Protocols
DOI 10.1038/s41596-021-00505-5PMC PMC8710232
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Abstract

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies. This protocol describes a two-phase assessment of senescence in cultured cells or clinical specimens by first identifying senescent cells using a combination of markers and then defining the senescent subtype by measuring pro-inflammatory activity.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper presents a two-phase algorithmic protocol to validate and specify cellular senescence in experimental and clinical specimens by combining lysosomal, proliferative, gene-expression, and SASP markers. It aims to reduce false positives/negatives from single markers and to identify pro-inflammatory senescent subtypes for basic and clinical senotherapy studies.

Key findings

  • The protocol uses Phase 1 to validate senescence via SA-β-Gal and/or LF with proliferation markers and core senescence mRNAs/proteins, and Phase 2 to specify the pro-inflammatory phenotype via six SASP factors: IL1A/IL6/IL8/IL1B/CXCL1/CXCL2.
  • In BJ fibroblasts treated with doxorubicin, >90% were EdU−SA-β-Gal+; vehicle-treated or untreated fibroblasts showed >90% EdU+SA-β-Gal−; untreated melanocytes showed only 30% EdU+SA-β-Gal−. In doxorubicin-treated mouse kidneys, ~15% of cells were SA-β-Gal positive; in cisplatin-treated mouse kidneys, a maximum of 4% were positive for p21 staining.
  • GL13/LF staining detected 50–65% positivity in senescent cells in culture, with 35–55% simultaneously positive for nuclear p16INK4A or p21WAF1/Cip1 and GL13; animal model tissues showed 8–26% senescent cells, and human primary non-treated classical Hodgkin lymphomas showed 1–34%.
  • A SASP panel (GDF15, MMP1, STC1, TIMP1, TIMP2) was measured in conditioned medium or human plasma, with expected higher levels in older individuals compared with younger individuals.

Methods

  • Protocol paper describing a two-phase algorithmic assessment for cultured cells and in vivo tissues; fresh biopsies are split for OCT freezing, fixation, and RNA extraction, while FFPE samples allow LF plus indirect markers.
  • Assays include SA-β-Gal at pH 6, EdU/Ki-67 proliferation staining, GL13 LF staining, RT-qPCR for p16INK4a, p21WAF1/Cip1, lamin B1, core senescence genes, and SASP genes; plasma collection follows CPTAC guidelines, and conditioned medium is collected serum-free and phenol red-free.
  • Analysis quantifies EdU/SA-β-Gal percentages from at least 300 cells in three independent experiments; RT-qPCR uses ΔCt/ΔΔCt with at least two housekeeping genes; SASP proteins are measured by mass spectrometry, ELISA, western blot, antibody arrays, or aptamer-based assays.

Limitations

  • Stated: combining all markers in the same sample is not yet possible without potential false positives or negatives; tissue SA-β-Gal analysis requires rapid freezing and can limit further histological analysis; GL13 is diluted in 70% ethanol and is incompatible with living cells; antigen retrieval complicates combining SA-β-Gal with additional markers.
  • Stated: senescence subtype identification remains limited because individual senescent cells cannot currently be isolated and purified from human tissues; the two-phase protocol is more complicated and time-consuming than many others, which may be a barrier to wide application.
  • Evident from the text: no universal or specific senescence marker exists, so multiple markers are required; SASP composition is highly heterogeneous and context-dependent.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41596-021-00505-5

Cites

78 of 78 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 50 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    A 'core' senescence signature exists: a set of genes commonly differentially expressed in all senescent cells analyzed.

    “However, these analyses allowed the identification of a ‘core’ senescence signature—a set of genes that are commonly differentially expressed in all the senescent cells analyzed5,38.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Hernandez-Segura, A. et al. Unmasking transcriptional heterogeneity in senescent cells. Curr. Biol. 27, 2652–2660.e4 (2017). source

      Not checked in the free run.

    • cites [38] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). source

      Not checked in the free run.

  • Findingown data

    Neither p21WAF1/Cip1 nor p16INK4a are consistently upregulated across different types of senescent cells.

    “Accordingly, by comparing whole-transcriptome datasets obtained from different types of senescent cells, we and others have recently shown that neither p21WAF1/Cip1 nor p16INK4a are consistently upregulated.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Hernandez-Segura, A. et al. Unmasking transcriptional heterogeneity in senescent cells. Curr. Biol. 27, 2652–2660.e4 (2017). source

      Not checked in the free run.

    • cites [38] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). source

      Not checked in the free run.

  • Findingcitation

    Analysis and comparison of whole-transcriptome datasets allowed definition of gene sets that associate specifically with individual senescent subtypes.

    “Importantly, the analysis and comparison of whole-transcriptome datasets allowed the definition of gene sets that associate specifically with individual senescent subtypes5,38.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Hernandez-Segura, A. et al. Unmasking transcriptional heterogeneity in senescent cells. Curr. Biol. 27, 2652–2660.e4 (2017). source

      Not checked in the free run.

    • cites [38] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). source

      Not checked in the free run.

  • Findingcitation

    A core set of SASP proteins are commonly produced and secreted by different types of senescent cells.

    “Recently, we have used mass spectrometry in cell culture and human plasma and identified a core set of SASP proteins that are commonly produced and secreted by different types of senescent cells38.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). source

      Not checked in the free run.

  • Findingown data

    In senescent cells in culture, 50-65% of the population is positive for GL13 staining.

    “In senescent cells in culture, 50–65% of the population is positive for GL13 staining, with 35–55% simultaneously positive for nuclear p16INK4A or p21WAF1/Cip1 and GL13 (Extended Data Fig. 1).”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 58
  • The quote only partly states this claim. 2
  • May combine two claims. 23

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In hybrid cytochemistry/immunofluorescence with NBT/BCIP substrate, the dark blue/purple signal develops after 5-7 min at room temperature.

    “Monitor the staining reaction under the light microscope until detection of the dark blue/purple signal (5–7 min) at RT.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    The GL13 staining reaction produces variably sized blue-black or brown cytoplasmic granules after an average time of 5-10 min.

    “Monitor the staining reaction under the microscope until detection of the signal (variably sized blue-black or brown cytoplasmic granules after an average time of 5–10 min).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Findingnone given

    In double immunocytochemistry and hybrid cytochemistry/immunocytochemistry, the dark brown immunocytochemical signal develops after an average time of 30-60 s at room temperature.

    “Monitor the staining reaction under the light microscope until detection of the dark brown signal (average time 30–60 s) at RT.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Fixed cells can be stored at 4 °C for up to 1 week.

    “Fixed cells can be stored at 4 °C for up to 1 week.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The GL13 dye can be stored for a maximum of 2-3 months at room temperature in the dark.

    “The dye can be stored for a maximum of 2–3 months at RT in the dark.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In vehicle-treated or untreated fibroblasts, more than 90% of cells are EdU+SA-β-Gal−.

    “In vehicle-treated or untreated fibroblasts, we can observe >90% EdU+SA-β-Gal−cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In human primary (non-treated) classical Hodgkin lymphomas, the range of senescent cells is 1-34% of total cells.

    “Lower percentages of senescent cells are expected in tissues from animal models (range of senescent cells: 8–26% of total cells – see figure legend of Extended Data Fig. 2 for details) and human primary (non-treated) classical Hodgkin lymphomas (range of senescent cells: 1–34% of total cells) (Extended Data Fig. 3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In the kidneys of doxorubicin-treated mice, approximately 15% of cells are positive for SA-β-Gal.

    “Moreover, in the kidneys of doxorubicin-treated mice, we can observe that ~15% of the cells are positive for SA-β-Gal.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    In BJ foreskin fibroblasts treated with doxorubicin, more than 90% of cells are EdU−SA-β-Gal+ (senescent).

    “In BJ foreskin fibroblasts treated with doxorubicin, we can observe >90% EdU−SA-β-Gal+ (senescent) cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    In the kidneys of cisplatin-treated mice, a maximum of 4% of cells are positive for p21 staining.

    “In the kidneys of cisplatin-treated mice, a maximum of 4% of the cells are positive for p21 staining.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In tissues from animal models, the range of senescent cells is 8-26% of total cells.

    “Lower percentages of senescent cells are expected in tissues from animal models (range of senescent cells: 8–26% of total cells – see figure legend of Extended Data Fig. 2 for details) and human primary (non-treated) classical Hodgkin lymphomas (range of senescent cells: 1–34% of total cells) (Extended Data Fig. 3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Additional stress conditions such as long-term confluence and prolonged serum starvation can trigger SA-β-Gal activity.

    “Moreover, additional stress conditions such as long-term confluence and prolonged serum starvation can trigger SA-β-Gal activity23.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Yang, N. & Hu, M. The limitations and validities of senescence associated-β-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells. Exp. Gerontol. 40, 813–819 (2005). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    p21WAF1/Cip1 is involved in DNA damage signaling and can be expressed by non-senescent cells upon DNA damage.

    “p21WAF1/Cip1 is also involved in DNA damage signaling34,35, and thus can be expressed by non-senescent cells upon DNA damage.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] Alcorta, D. A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. USA 93, 13742–13747 (1996). source

      Not checked in the free run.

    • cites [35] Karimian, A., Ahmadi, Y. & Yousefi, B. Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair 42, 63–71 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    p16INK4a is frequently expressed late after senescence induction, while expression of p21WAF1/Cip1 occurs earlier.

    “p16INK4a is frequently expressed late after senescence induction, while expression of p21WAF1/Cip1, a CDK4/6 and CDK2 inhibitor, occurs earlier34.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Alcorta, D. A. et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. USA 93, 13742–13747 (1996). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Developmental senescent cells express p21WAF1/Cip1 but not p16INK4a, while the reverse is observed in melanocytic nevi.

    “For example, developmental senescent cells express p21WAF1/Cip1 but not p16INK4a9,10, while the reverse is observed in melanocytic nevi36,37.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [9] Muñoz-Espín, D. et al. Programmed cell senescence during mammalian embryonic development. Cell 155, 1104 (2013). source

      Not checked in the free run.

    • cites [10] Storer, M. et al. Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 155, 1119 (2013). source

      Not checked in the free run.

    • cites [36] Michaloglou, C. et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005). source

      Not checked in the free run.

    • cites [37] Gray-Schopfer, V. C. et al. Cellular senescence in naevi and immortalisation in melanoma: a role for p16? Br. J. Cancer 95, 496–505 (2006). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    SA-β-Gal can also be detected in macrophages, hair follicles and sebaceous glands.

    “However, it can also be detected in macrophages22, as well as in hair follicles and sebaceous glands21.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22] Hall, B. M. et al. p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli. Aging 9, 1867–1884 (2017). source

      Not checked in the free run.

    • cites [21] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Senescent cells are generally characterized by increased lysosomal content occurring as a consequence of defective lysosomal turnover or of multi-faceted changes in autophagy.

    “Senescent cells are generally characterized by increased lysosomal content occurring as a consequence of defective lysosomal turnover24 or of multi-faceted changes in autophagy25.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [24] Hernandez-Segura, A., Nehme, J. & Demaria, M. Hallmarks of cellular senescence. Trends Cell Biol. 28, 436–453 (2018). source

      Not checked in the free run.

    • cites [25] Kang, C. & Elledge, S. J. How autophagy both activates and inhibits cellular senescence. Autophagy 12, 898–899 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    LF accumulation in aged post-mitotic tissues occurs most probably in the context of post-mitotic cellular senescence (PoMiCS), which is suspected to play a significant role in driving tissue aging, but LF can be present in non-senescent post-mitotic tissues and thus is not strictly senescence-specific.

    “Of note, LF accumulation in aged post-mitotic tissues occurs most probably in the context of post-mitotic cellular senescence (PoMiCS), which is suspected to play a significant role in driving tissue aging29,30, but can be present in non-senescent post-mitotic tissues and thus is not strictly senescence-specific31.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [29] von Zglinicki, T., Wan, T. & Miwa, S. Senescence in post-mitotic cells: a driver of aging? Antioxid. Redox Signal. https://doi.org/10.1089/ars.2020.8048 (2020). source

      Not checked in the free run.

    • cites [30] Sapieha, P. & Mallette, F. A. Cellular senescence in postmitotic cells: beyond growth arrest. Trends Cell Biol. 28, 595–607 (2018). source

      Not checked in the free run.

    • cites [31] Brunk, U. T. & Terman, A. Lipofuscin: mechanisms of age-related accumulation and influence on cell function. Free Radic. Biol. Med. 33, 611–619 (2002). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    SA-β-Gal is active in most senescent cells and is absent in the majority of proliferating and quiescent cells.

    “SA-β-Gal is active in most senescent cells and is absent in the majority of proliferating and quiescent cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    All samples should be measured in duplicate.

    “All samples should be measured in duplicate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductreasoning

    Mycoplasma contamination will invalidate experiments that analyze the SASP through either mRNA or proteomic analysis.

    “Contamination will invalidate experiments that analyze the SASP through either mRNA or proteomic analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductreasoning

    The SA-β-gal staining assay must be done as soon as possible after the frozen tissue has been sectioned because the activity of the enzyme is quickly lost upon exposure to higher temperatures.

    “Moreover, it is critical that the SA-β-gal staining assay is done as soon as possible after the frozen tissue has been sectioned, as the activity of the enzyme is quickly lost upon exposure to higher temperatures.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    The combination of EdU and SA-β-gal staining for tissues remains unreliable.

    “EdU incorporation in vivo can be done in actively proliferating organs (e.g., intestine), but the combination of EdU and SA-β-gal staining for tissues remains unreliable and will not be covered in this protocol.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Because of the lack of assays for absolute quantification of most mRNAs, control samples are important to determine relative fold changes.

    “Because of the lack of assays for the absolute quantification of most of the mRNAs measured, it is important that control samples (e.g., non-senescent cells, healthy tissue) are included in the analysis to determine relative fold changes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationnone given

    The tools necessary for the algorithmic approach are easy to obtain and the methods do not require special equipment, thus remaining highly accessible to most laboratories.

    “Importantly, and in contrast to other multi-marker approaches, the tools necessary for our algorithmic approach are easy to obtain and the methods do not require special equipment, thus remaining highly accessible to most laboratories.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    SASP protein components are identified based on significantly changed abundance in the conditioned medium of senescent cells versus non-senescent control cells.

    “SASP protein components are identified based on significantly changed abundance in the conditioned medium of senescent cells versus non-senescent control cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Identification of senescence subtypes remains limited mainly because it is currently impossible to isolate and purify individual senescent cells from human tissues.

    “Fifth, the identification of senescence subtypes remains limited, mainly because it is currently impossible to isolate and purify individual senescent cells from human tissues.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Positive controls, negative controls and technical negative controls should always be utilized for LF staining.

    “Positive controls (samples with established senescence), negative controls (samples with absence of senescence) and technical negative controls (samples in which key reagents have been omitted during the staining procedure) should always be utilized17.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Thompson, P. J. et al. Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes. Cell Metab. 29, 1045–1060.e10 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    LF staining has the advantage of remaining detectable in paraffin-embedded material and is thus applicable in archival FFPE material.

    “Another readout of enhanced lysosomal activity in senescent cells is staining for LF, which has the advantage of remaining detectable in paraffin-embedded material and is thus applicable in archival (FFPE) material.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    In both preclinical and clinical settings, defining a senescent cell on the basis of individual markers can under- or overestimate the number of senescent cells.

    “In both preclinical and clinical settings, it is common to define a senescent cell on the basis of individual markers which can under- or overestimate the number of senescent cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    Proper cell culture maintenance is required to ensure in vitro results generated in the protocol are valid.

    “Proper cell culture maintenance is required to ensure in vitro results generated in our protocol are valid.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    It remains essential to use a panel of core SASP proteins for validation of the senescent phenotype.

    “Since none of the individual SASP proteins are senescence-specific, it remains essential to use a panel of core SASP proteins for validation of the senescent phenotype.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Combining all the markers in the same sample is not yet possible without incurring potential false positives or negatives.

    “First, combining all the markers in the same sample is not yet possible without incurring potential false positives or negatives.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    At least two housekeeping genes should be used for RT-qPCR in line with MIQE.

    “In line with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments), we recommend using at least two housekeeping genes48,49.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [48] Bustin, S. A. et al. The need for transparency and good practices in the qPCR literature. Nat. Methods 10, 1063–1067 (2013). source

      Not checked in the free run.

    • cites [49] Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductcitation

    GL13 is diluted in 70% (vol/vol) ethanol, rendering this assay inconsistent with analysis of living cells.

    “Third, GL13 is diluted in 70% (vol/vol) ethanol, rendering this assay inconsistent with analysis of living cells17.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Thompson, P. J. et al. Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes. Cell Metab. 29, 1045–1060.e10 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Culture medium used during the 24-h collection step must be free of serum and as many other protein components/contaminants as possible.

    “▲CRITICAL STEP Culture medium used during this step must be free of serum and as many other protein components/contaminants as possible.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For each gene, a sample containing RNase-free water instead of cDNA should always be included to ensure amplified products are not primer dimers or from contamination.

    “▲CRITICAL STEP For each gene, always include a sample containing Rnase-free water instead of cDNA to ensure amplified products are not primer dimers or from contamination.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    Tissue analysis for SA-β-gal is limited unless the sample is quickly frozen and analyzed to maintain enzymatic activity, which can limit further histological analysis due to poor tissue structure preservation.

    “Second, tissue analysis for SA-β-gal is limited unless the sample is quickly frozen and analyzed to maintain enzymatic activity, which can limit further histological analysis due to poor tissue structure preservation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Determination of optimal dilution and incubation time of the secondary antibody is crucial to avoid nonspecific staining.

    “Determination of optimal dilution and incubation time of the secondary antibody to avoid nonspecific staining is crucial.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Recording cell counts for every flask is essential for accurate downstream protein quantification.

    “▲CRITICAL STEP Recording cell counts for every flask is essential for accurate downstream protein quantification.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Ki-67 immunofluorescence staining should be done using slides adjacent to the slides used for SA-β-galactosidase staining.

    “▲CRITICAL The staining should be done using slides adjacent to the slides used for SA-β-galactosidase staining.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Suitable house-keeping genes are critical for normalization.

    “It is also critical that suitable ‘house-keeping’ genes are used for normalization47.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [47] Hernandez-Segura, A., Rubingh, R. & Demaria, M. Identification of stable senescence-associated reference genes. Aging Cell 18, e12911 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The SASP is highly complex and heterogeneous, with a composition influenced by many intrinsic and extrinsic factors.

    “The SASP is highly complex and heterogeneous, with a composition that is influenced by many intrinsic and extrinsic factors5,38.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Hernandez-Segura, A. et al. Unmasking transcriptional heterogeneity in senescent cells. Curr. Biol. 27, 2652–2660.e4 (2017). source

      Not checked in the free run.

    • cites [38] Basisty, N. et al. A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol. 18, e3000599 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    At least two housekeeping genes should be used for reference in RT-qPCR, in line with MIQE guidelines.

    “In line with MIQE guidelines, we recommend using at least two housekeeping genes for reference.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites MIQE guidelines

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). The quote only partly states this claim.

    Study design and conductnone given

    The second and third washes with 50% ethanol are essential for removal of nonspecific staining.

    “The second and third washes are essential for removal of nonspecific staining.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    Cells should be washed thoroughly but gently to remove residual proteins derived from serum and other media supplements.

    “▲CRITICAL STEP Wash cells thoroughly but gently in order to remove residual proteins derived from serum and other media supplements.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Incubation time for SA-β-Gal staining needs to be optimized so that staining is still negative in the control group but positive in treated groups.

    “▲CRITICAL STEP Incubation time needs to be optimized so that staining is still negative in the control group, but positive in treated groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Phenol red can interfere with protein quantification during the procedure.

    “Phenol red can also interfere with protein quantification during the procedure.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisereasoning

    Abundant exogenous protein contamination can severely limit the identification and quantification of true secreted proteins in proteomic analyses due to dynamic range problems and ionization suppression.

    “Abundant exogenous protein contamination can severely limit the identification and quantification of true secreted proteins in the proteomic analyses (due to potential dynamic range problems and ionization suppression).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductcitation

    Confluent cell populations are unable to replicate and are often positive for SA-β-gal activity.

    “Confluent cell populations should be avoided as they are unable to replicate and are often positive for SA-β-gal activity21.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Dimri, G. P. et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    If the culture medium must contain protein components, these proteins must be excluded from later proteomic data analysis.

    “If the culture medium must contain protein components, these proteins must be excluded from later proteomic data analysis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Study design and conductnone given

    Co-staining procedures are difficult and produce a significant amount of artifactual false positives and negatives.

    “However, because co-staining procedures are difficult and produce a significant amount of artifactual false positives and negatives, we recommend a sequential staining (i.e., independent staining on sequential and adjacent sections) for frozen in vivo samples without pre-fixation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    Levels of multiple transcripts should be measured at the same time and in the same sample to avoid false positives.

    “Since none of these genes appear to be uniquely expressed by senescent cells, the levels of multiple transcripts should be measured at the same time and in the same sample to avoid false positives.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The species of the animal serum should be the same as the species of the secondary antibody.

    “▲CRITICAL STEP The species of the animal serum should be the same as the species of the secondary antibody.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The sectioned slides should be kept at −20 °C until all sectioning is done.

    “▲CRITICAL The sectioned slides should also be kept at −20 °C until all sectioning is done.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    DDR proteins such as γ-H2AX and 53BP1 are often present in senescent cells, but these markers also appear in non-senescent cells with transient DNA damage.

    “DDR proteins such as γ-H2AX and 53BP1 are often present in senescent cells, but these markers also appear in non-senescent cells with transient DNA damage124.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [124]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The choice of the right reference gene depends on the experimental system and requires direct testing by the investigator.

    “but the choice of the right reference will depend upon the experimental system and requires direct testing by the investigator.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Both senescent and non-senescent (control) secreted protein samples should be prepared for each senescence condition tested in multiple biological replicates (at least three).

    “▲CRITICAL Prepare both senescent and non-senescent (control) secreted protein samples for each senescence condition tested in multiple biological replicates (at least three).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The geometric mean of β-actin and α-tubulin can be used as a general reference combination.

    “The geometric mean of β-actin and α-tubulin can be used as a general combination”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The two-phase protocol is more complicated than many others, especially for studies not focused on cellular senescence, which might be a barrier for wide application.

    “Sixth, our two-phase protocol is more complicated than many others, especially for studies not focused on cellular senescence, which might be a barrier for wide application.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    The staining should be done immediately after sectioning; within 1 h is recommended.

    “▲CRITICAL The staining should be done immediately after sectioning; within 1 h is recommended.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A core senescence signature was previously identified based on fold change in expression compared to non-senescent tissues or cells and p-values > 0.05.

    “a subset of genes that are part of the core senescence signature we identified previously on the basis of by fold change in expression compared to non-senescent tissues or cells and p-values > 0.05)5”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Hernandez-Segura, A. et al. Unmasking transcriptional heterogeneity in senescent cells. Curr. Biol. 27, 2652–2660.e4 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Results of tissues should be compared versus their appropriate control for the treatment used.

    “Compare results of tissues versus their appropriate control for the treatment used.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The two-phase algorithmic protocol can minimize the risk of under- or overestimating the number of senescent cells and provide preliminary information on potential senescence-associated pathological functions.

    “The protocol, while being more time-consuming than the ones currently in use40-43, can minimize the risk of under- or overestimating the number of senescent cells, and also provide preliminary information on potential senescence-associated pathological functions.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    RNA must always be kept on ice to prevent degradation, and equipment must be kept RNase-free by cleaning with RNase AWAY.

    “▲CRITICAL STEP Ensure that RNA is always kept on ice to prevent degradation and equipment is kept RNAse free by cleaning with RNase AWAY.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    During proteomic analysis, secreted protein levels are normalized to cell counts.

    “During proteomic analysis, secreted protein levels are normalized to cell counts.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    Antigen retrieval is necessary for many antibodies targeting other senescence markers, making it difficult to combine SA-β-gal with additional markers.

    “Fourth, antigen retrieval is necessary for many antibodies targeting other senescence markers (e.g., p21WAF1/Cip1), which is more easily performed in paraffin-embedded samples, making it difficult to combine SA-β-gal with additional markers.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Study design and conductnone given

    The presence of serum can compromise the detection of SASP proteins secreted from cells.

    “This is important as the presence of serum can compromise the detection of SASP proteins secreted from cells.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    Use the recommended dilution of the antibody specified on the datasheet; if not specified, the typical starting dilution can be 2-5 μg/mL.

    “▲CRITICAL STEP Use the recommended dilution of the antibody specified on the datasheet. If not specified, the typical starting dilution can be 2–5 μg/mL.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Study design and conductnone given

    For in vivo samples, senescence validation entails double-staining for SA-β-Gal and Ki-67 in sequential sections, co-staining of LF and Ki-67, sole staining of p21WAF1/Cip1, RT-PCR analyses for p16INK4a and p21WAF1/Cip1 mRNAs, and measurements of core SASP secreted proteins in human plasma.

    “▲CRITICAL For in vivo samples (Steps 53–115), senescence validation entails double-staining for SA-β-Gal and Ki-67 in sequential sections, co-staining of LF and Ki-67 and sole staining of p21WAF1/Cip1, RT-PCR analyses for p16INK4a and p21WAF1/Cip1 mRNAs, as well as measurements of core SASP secreted proteins in human plasma.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These factors are commonly described as the mediator of the pro-disease functions of senescent cells in vivo.

    “These factors are commonly described as the mediator of the pro-disease functions of senescent cells in vivo59.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [59] Laberge, R. M. et al. MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nat. Cell Biol. 17, 1049–1061 (2015). source

      Not checked in the free run.

71 of 71

  • The paper relies on this without support.

    Many senescence-inducing stresses activate a persistent DNA damage response and most induce expression of cell cycle inhibitors, most frequently p53/p21WAF1/Cip1 and p16INK4a/RB1.

    “Many of these stresses activate a persistent DNA damage response (DDR) and most induce expression of cell cycle inhibitors, most frequently p53/p21WAF1/Cip1 and p16INK4a/RB1.”

  • The paper relies on this without support.

    The protocol can be adapted for use in other species with minor adjustments.

    “We provide details to analyze human specimens, but with minor adjustments the protocol can be adapted for use in other species.”

  • The paper relies on this without support.

    The selected panel of pro-inflammatory factors can specify the subtypes of senescent cells.

    “In phase 2, we propose to measure a panel of pro-inflammatory factors—also evaluated at the mRNA level—in order to specify the subtypes of senescent cells.”

  • The paper relies on this without support.

    CO2 affects the pH of the SA-β-gal staining solution and will artifactually modify the results.

    “CO2 affects the pH and will artifactually modify the results.”

  • The paper relies on this without support.

    Evaporation of the SA-β-gal staining solution leads to the formation of crystals.

    “Evaporation leads to the formation of crystals.”

  • The paper relies on this without support.

    SA-β-Gal positive cells present a blue perinuclear staining.

    “Positive cells present a blue perinuclear staining.”

  • The paper relies on this without support.

    Evaporation of ethanol leads to the formation of crystals, and such crystals indicate that the GL13 reagent is not completely dissolved.

    “Evaporation of ethanol leads to the formation of crystals. The latter indicates the reagent is not completely dissolved.”

  • The paper relies on this without support.

    Using primary antibodies raised in different species avoids cross-reactivity in co-staining.

    “Primary antibodies should be raised in different species.”

  • The paper relies on this without support.

    Using secondary antibodies conjugated with two different fluorochromes allows separate detection of the two targets.

    “Use secondary antibodies conjugated with two different fluorochromes.”

  • The paper relies on this without support.

    The composition of the SASP can vary dramatically depending on the length of time after senescence induction.

    “▲CRITICAL The composition of the SASP can vary dramatically depending on the length of time after senescence induction.”

  • The paper relies on this without support.

    Phenol red in the medium at this step can interfere with quantification of protein content by BCA assay in subsequent steps.

    “▲CRITICAL STEP Phenol red in the medium at this step can interfere with quantification of protein content by BCA assay in subsequent steps.”

  • The paper relies on this without support.

    Overfixation (beyond 24 h) can influence the staining results.

    “▲CRITICAL STEP Avoid overfixation (limit 24 h), which can influence the staining results.”

  • The paper relies on this without support.

    Covering with a coverslip is crucial to avoid evaporation of ethanol during GL13 staining.

    “▲CRITICAL STEP Covering with the coverslip is crucial to avoid evaporation of ethanol and should be performed in a chamber.”

  • The paper relies on this without support.

    Repetition of the GL13 staining procedure can enhance the reaction, especially when overfixation of the tissue has taken place.

    “▲CRITICAL STEP Upon weak signal, repetition of the staining procedure with the GL13 solution can result in enhancement of the reaction especially when overfixation of the tissue has taken place.”

  • The paper relies on this without support.

    Additional washes are required for removal of nonspecific staining.

    “▲CRITICAL STEP The additional washes are required for removal of nonspecific staining.”

  • The paper relies on this without support.

    Determining the optimal dilution and incubation time of the secondary antibody is crucial to avoid nonspecific staining.

    “▲CRITICAL STEP It is crucial to determine the optimal dilution and incubation time of the secondary antibody to avoid nonspecific staining.”

  • The paper relies on this without support.

    Keeping tissue on dry ice and not allowing it to thaw prevents endogenous RNAse activation.

    “▲CRITICAL STEP Ensure the tissue is kept on dry ice and is not allowed to thaw, to prevent endogenous RNAse activation.”

  • The paper relies on this without support.

    Preventing liquid nitrogen evaporation during grinding prevents the sample from thawing.

    “▲CRITICAL STEP Ensure the liquid nitrogen does not evaporate while the tissue is being ground to prevent the sample from thawing. Add more liquid nitrogen if needed and continue grinding.”

  • The paper relies on this without support.

    Ground tissue powder can be stored at −80 °C for up to 1 year.

    “■PAUSE POINT Ground tissue powder can be stored at −80 °C for up to 1 year.”

  • The paper relies on this without support.

    Mounted slides can be stored at RT for at least 1 month.

    “■PAUSE POINT Mounted slides can be stored at RT for at least 1 month.”
20 of 20

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Senescence is heterogeneous: a variety of insults may incite senescence, the inflammatory secretory phenotype is modified depending on cell type, and the senescent phenotype may change based on the stage of senescence.

    “A challenge in targeting senescence is the apparent heterogeneity of this cellular state, wherein a variety of insults may incite senescence, the inflammatory secretory phenotype is modified depending on cell type, and the senescent phenotype may even change based on the stage of senescence [22–24].”

    From Untangling senescent and damage‐associated microglia in the aging and diseased brain (2023) · cites this paper as [24]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • In vitro phase is said to end at Step 49 but includes Steps 50-52

    Minor

    The stated Step 1-49 range excludes three steps of the conditioned-medium protein analysis that the same passage includes in phase 1.

  • Plasma protein measurement points to the transcript-measurement step

    Minor

    Step 117 directs readers measuring proteins in plasma to Step 116, which describes measuring mRNA by RT-PCR rather than plasma proteins.

2 of 2

Cites

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Authors
Kohli, J., Wang, B., Brandenburg, S. M., Basisty, N., Evangelou, K., Varela-Eirin, M., Campisi, J., Schilling, B., Gorgoulis, V., & Demaria, M.
Affiliations
  • University Medical Center Groningen (NL)
  • Buck Institute for Research on Aging (US)
  • National and Kapodistrian University of Athens (GR)
Corresponding author
  • Gorgoulis, V.
  • Demaria, M.
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2021-04-28
Volume
16
Issue
5
Pages
2471-2498
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
DOI
10.1038/s41596-021-00505-5
PMC
PMC8710232
OpenAlex
W3158340771
Semantic Scholar
c6b0a7b0d9490a30e38c5f113a0e0b3b25638d3f
Cited by
196
References
78
Citations by year
2026: 25 · 2025: 41 · 2024: 47 · 2023: 35 · 2022: 33 · 2021: 15
FWCI
20.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, other-oa)
  • PubMed Central (acceptedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, other-oa)
Funders
  • KWF Kankerbestrijding (10989)
  • EC | Horizon 2020 Framework Programme
  • U.S. Department of Health & Human Services | NIH | National Institute on Aging
Topics
  • Health Sciences › Medicine › Physiology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
cellular senescence, senescence-associated phenotype, senescence markers, senescence-associated genes, pro-inflammatory markers, senotherapy, algorithmic assessment
MeSH terms
Algorithms, Cytological Techniques, Humans, Lysosomes, Biomarkers, Cellular Senescence, Cell Proliferation

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

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