Measuring the Signals Released by Aging Cells

Quantitative reverse transcription PCR (qRT-PCR) is one of the most widely used molecular biology techniques to measure gene expression. qRT-PCR involves two primary steps: the reverse transcription of RNA into complementary DNA (cDNA) using reverse transcriptase, followed by amplification of specific gene targets using polymerase chain reaction with fluorescent detection. The real-time monitoring of fluorescence, typically via SYBR Green or TaqMan probes, allows quantification of transcript abundance in relative or absolute terms [52]. In the context of senescence research, it is particularly valuable for quantifying the transcriptional activation of SASP components such as pro-inflammatory cytokines, chemokines, growth factors, and matrix remodeling enzymes. The technique offers high sensitivity, specificity, and quantitative accuracy, making it ideal for studying SASP gene dynamics in vitro and in vivo settings.

In senescence studies, qRT-PCR has been instrumental in establishing the SASP as a defining feature of various senescence models. For example, Kuilman et al. used qRT-PCR to demonstrate increased mRNA levels of IL6 and IL8 in human fibroblasts undergoing oncogene-induced senescence [35]. Similarly, Rodier et al. utilized qRT-PCR to link persistent DNA damage signaling to sustained expression of IL-6, IL-1A, and other SASP factors [53].

qRT-PCR is also employed to monitor changes in SASP gene expression following pharmacological or genetic interventions. For instance, Laberge et al. used this method to show that inhibition of the mTOR pathway via rapamycin leads to a significant downregulation of IL-1A, IL-6, and other SASP-related transcripts, linking translational control to SASP modulation [36]. qRT-PCR is particularly advantageous when analyzing samples with limited RNA quantity, such as sorted cell populations or small tissue biopsies. The use of specific primers allows targeted quantification of key SASP components, making it a reliable tool for senescence diagnostics. Moreover, advancements in high-throughput qRT-PCR platforms now allow for simultaneous measurement of dozens to hundreds of SASP-related genes across large sample cohorts, facilitating comparative and longitudinal studies [54].

Despite its utility, qRT-PCR has limitations. It measures transcript levels, which may not directly correlate with protein abundance due to post-transcriptional regulation. Therefore, discrepancies between mRNA and protein levels may be observed in certain senescent cell models. The SASP, in its earliest characterization, encompassed roughly fifty biomolecules—including cytokines, chemokines (CXCLs), growth factors, and proteolytic enzymes. These were predominantly identified using biased methodologies such as antibody-based arrays or targeted transcriptional profiling approaches. Although such techniques have significantly advanced our understanding of the senescent cell phenotype, they inherently provide a partial view. This is where proteomic analysis emerges as a powerful counterpart.

An alternative that could offer methodological advantages to qPCR is digital PCR (dPCR). By partitioning a sample into thousands to millions of microreactions, dPCR enables absolute quantification of nucleic acid targets without the need for standard curves, thereby eliminating dependence on amplification efficiency assumptions. This partitioning also enhances sensitivity for detecting rare targets and increases tolerance to PCR inhibitors, as their effects are diluted among partitions. Furthermore, dPCR provides superior precision and reproducibility at low template copy numbers, making it particularly suitable for applications requiring high analytical accuracy. However, dPCR also has notable limitations. Its dynamic range is narrower than that of qPCR, as high template concentrations can saturate partitions, necessitating prior dilution. Throughput is generally lower, and assay times can be longer due to additional partitioning and detection steps. Moreover, the technique requires specialized instrumentation and consumables, leading to higher per-sample costs [55,56].

RNA sequencing (RNA-seq) has emerged as a transformative technology in transcriptomic analysis, enabling unbiased, high-throughput quantification of gene expression across the entire transcriptome. It involves the extraction of total or polyadenylated RNA, followed by fragmentation, reverse transcription into cDNA, library preparation, and high-throughput sequencing. The resulting sequences are aligned to a reference genome or transcriptome to quantify gene expression levels [57].

In senescence research, RNA-seq has an important role for dissecting the complexity and heterogeneity of the SASP, especially in contexts where its composition is variable or unknown. Unlike targeted approaches such as qRT-PCR, RNA-seq does not rely on prior knowledge of gene sequences, thus offering a comprehensive view of transcriptional changes that occur during cellular senescence. This approach allows not only the detection of differentially expressed genes but also the discovery of novel transcripts, splice variants, and non-coding RNAs that may contribute to SASP regulation.

A landmark example of RNA-seq application in SASP profiling comes from Basisty et al., who generated a detailed "SASP Atlas" by performing RNA-seq and proteomics across multiple human cell types, including fibroblasts, endothelial cells, and preadipocytes, undergoing various senescence triggers (e.g., ionizing radiation, replicative exhaustion, oncogene activation). The study revealed both core SASP factors (e.g., IL-6, IL-8, MMP-1) and stimulus-specific components, underscoring the heterogeneity of the SASP and the importance of cell type and senescence inducer in shaping secretory profiles [58].

Other studies have leveraged RNA-seq to identify SASP expression patterns in specific tissues or disease models. For example, Casella et al. used RNA-seq to compare senescence profiles of in human fibroblasts, human umbilical vein endothelial cells, and human alveolar endothelial cells through different triggers of senescence [37]. RNA-seq is also instrumental in identifying transcriptional regulators of the SASP. In Herranz et al., RNA-seq was applied to showcase that mTOR controls the SASP by regulating the translation of the MK2/MAPKAPK2 kinase through 4EBP1 [38]. Similarly, Hoare et al. used RNA-seq to study the effect of NOTCH1 activity on SASP through two distinct secretomes, one representing TGF-β and the other pro-inflammatory cytokines [59].

Single-cell RNA sequencing (scRNA-seq) can offer advantages over bulk RNA sequencing for the analysis of tissues affected by cellular senescence. Bulk RNA-seq measures average transcript abundance across all cells, thereby obscuring heterogeneity and potentially masking transcriptional profiles of rare senescent cells. In contrast, scRNA-seq enables the identification and characterization of individual senescent cells within complex tissues, revealing subpopulations that differ by senescence trigger, stage, or cell type. This resolution facilitates the detection of co-expression patterns involving canonical senescence markers, alongside SASP factors. Furthermore, integration of scRNA-seq with spatial transcriptomics can localize senescent cells within tissue architecture, while trajectory inference can elucidate transcriptional transitions toward senescence [60,61]. Limitations of its use could be higher cost, greater technical complexity, and reduced sensitivity for low-abundance transcripts.

A human aging study employed scRNA-seq data to characterize SASP features in the aging testis, identifying IGFBP7—expressed in Leydig cells—as a novel SASP-associated gene and potential biomarker of testicular aging [39]. Voight et al. employed scRNA-seq to characterize transcriptional diversity among senescent cells, showing that SASP expression varies markedly between individual cells even within a clonal population [62]. This variability may explain differences in SASP function in vivo and suggests that cellular context plays a major role in modulating secretory behavior.

Despite its strengths, RNA-seq has its limitations in SASP research. Transcript levels do not always correlate with protein abundance or activity due to post-transcriptional, translational, or secretion-level regulation. Moreover, the data complexity of RNA-seq requires robust bioinformatic pipelines for quality control, alignment, normalization, and statistical analysis. Tools such as DESeq2 and edgeR are commonly used for differential expression analysis [63]. When used in combination with complementary proteomic and functional analyses, they help create a multi-dimensional profile of the SASP that reflects both cellular state and biological impact.

RNA in situ hybridization (RNA-ISH) is a molecular technique used to detect specific RNA molecules (e.g., mRNAs, long non-coding RNAs, viral RNAs) directly within intact cells and tissues, preserving spatial context. It allows researchers to identify where a transcript is expressed (e.g., which cell types or tissue regions) and how much is being expressed at the level of individual cells. Synthetic RNA or DNA probes are designed to be complementary to a specific RNA sequence, and the labeled probe binds to the target RNA within fixed cells or tissue sections. The hybridized probes are visualized using chromogenic methods (color development using alkaline phosphatase or HRP) or fluorescent labels (multiplexed RNA-ISH, e.g., RNAscope, ViewRNA) [64,65].

Lorda-Diez et al. used RNA-ISH while studying cell senescence and DNA damage in the remodeling processes accounting for heart morphogenesis. They found intensified expression of secreted factors characteristic of the senescent secretome, such as pro-inflammatory cytokines (IL-1β, IL-6), growth factors (IGF1, IGFBP5, TGF-βs), and tissue remodeling factors (ADAMTS9, MMP-2, MMP-9) [66].

RNA-ISH also provides the ability to study novel non-coding RNAs. Recent studies have begun to uncover the role of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in the regulation of the SASP. For example, Lin et al. used RNA-ISH to identify lncRNA MALAT1 in gallbladder cancer cells, with their results suggesting that it potentiates growth and inhibits senescence by antagonizing ABI3BP in these cancer cells [67].

Thus, RNA in situ hybridization is a powerful, spatially resolved method for identifying senescent cells and their secretory phenotypes within tissues. It complements bulk RNA analysis and can link SASP gene expression to histological features—an especially valuable feature for aging, cancer, and degenerative disease research. Of course it has its limitations as it requires fixed tissue. In addition, there are difficulties in identifying targets that have low RNA copies, and it has lower throughput compared to transcriptomics [68].

Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a widely employed immunological technique used to quantitatively measure specific proteins in complex biological samples. ELISA operates on the principle of antibody-antigen specificity. A capture antibody is immobilized on a solid surface (typically a 96-well microplate), which selectively binds the target protein in the sample. A secondary detection antibody, conjugated to an enzyme such as horseradish peroxidase (HRP), binds to the captured antigen, forming a "sandwich" complex. Upon addition of a substrate (e.g., TMB), a measurable colorimetric change is produced, which is directly proportional to the concentration of the target analyte. The reaction is then quantified using a plate reader, with high sensitivity and reproducibility [40,69].

In the context of cellular senescence research, ELISA is a fundamental tool for detecting and quantifying soluble SASP components—cytokines, chemokines, growth factors, and matrix remodeling enzymes—in conditioned media, tissue lysates, plasma, and other bodily fluids. For instance, Coppé et al. utilized ELISA to measure elevated levels of IL-6 and IL-8 in the conditioned media of human fibroblasts undergoing oncogene-induced senescence (OIS) [70]. These findings complemented transcriptomic data and provided direct evidence of increased secretion of SASP factors. Similarly, Acosta et al. demonstrated via ELISA that IL-1α functions upstream of the IL-6/IL-8 axis in the SASP regulatory cascade, highlighting the assay's utility in dissecting cytokine hierarchies [71]. In the study by Wiley et al., it was used in conjunction with TMT-based MS to quantify secreted proteins from senescent and proliferating fibroblasts, revealing distinct proteomic signatures associated with different senescence triggers. Notably, the researchers discovered that while some proteins were consistently secreted regardless of senescence inducer (e.g., IL-8, MMP-1), others were unique to specific senescence pathways, illustrating the pathway-dependent nature of the SASP [72].

ELISA remains a gold-standard technique for the quantitative analysis of soluble SASP components, particularly in conditioned media and body fluids. Its simplicity, specificity, adaptability, and high sensitivity (typically in the picogram per milliliter range) make it indispensable for both mechanistic and translational studies of cellular senescence. However, it does have its limitations. It is a targeted assay, requiring prior knowledge of the proteins of interest and the availability of high-affinity, specific antibodies. Cross-reactivity and batch-to-batch variability in antibody performance can affect assay reliability. Moreover, ELISA cannot detect unknown or unexpected components of the SASP, nor does it provide information about post-translational modifications, which can modulate the function or activity of SASP proteins. Thus, it is most effective when used for quantitative validation of candidate SASP factors initially identified by unbiased transcriptomic or proteomic screening. When integrated with complementary approaches such as RNA-seq, qRT-PCR, and mass spectrometry, ELISA contributes to a robust, multi-level characterization of the SASP, facilitating both basic research and the development of senescence-targeted interventions [73,74].

Western Blotting

Western blotting is a classical and widely used protein detection technique that plays a critical role in senescence research by enabling the qualitative and semi-quantitative assessment of individual SASP components. While not as high-throughput as ELISA or mass spectrometry, Western blotting offers key advantages in specificity, validation of protein identity, and the detection of post-translational modifications (PTMs), making it useful for characterizing SASP regulators and effectors [41]. The technique involves the separation of often denatured proteins by molecular weight through SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), followed by transfer to a membrane (typically PVDF or nitrocellulose) and probing with specific primary and secondary antibodies. Detection is typically achieved through chemiluminescence or fluorescence, providing visual confirmation of protein presence and relative abundance [42,75].

In the context of SASP, Western blotting is commonly used to validate the presence of secreted or intracellular regulators such as IL-6, IL-1α, TNF-α, matrix metalloproteinases (e.g., MMP-1, MMP-3), and signaling proteins involved in SASP induction pathways, including components of the NF-κB, mTOR, and p38 MAPK signaling axes. For instance, Orjalo et al. used Western blotting to demonstrate that membrane-bound IL-1α, a key upstream regulator of SASP gene expression, is expressed at higher levels in senescent fibroblasts than in proliferating controls [76]. This study provided mechanistic insight into how IL-1α activates NF-κB signaling and perpetuates the SASP. Similarly, Herranz et al. used Western blotting to confirm changes in the phosphorylation state of mTOR in therapy-induced senescence, supporting a regulatory role of mTOR signaling in SASP protein production [38].

Western blotting is also used to monitor the intracellular accumulation of SASP-related transcription factors and modulators. For example, Rodier et al. demonstrated persistent DNA damage signaling in senescent cells by Western blot detection of ATM, NBS1, and CHK2, which are associated with the chronic DDR (DNA damage response) that sustains SASP gene expression [53]. Moreover, Western blotting is capable of detecting post-translational modifications—such as phosphorylation, acetylation, and ubiquitination—that critically regulate SASP protein activity and stability [77].

While Western blotting is often performed on cell lysates, it can also be applied to concentrated conditioned media to detect secreted SASP proteins. However, this requires protein concentration steps (e.g., ultrafiltration or TCA precipitation) and careful normalization due to variable protein yields in secretomes. Some studies, including Acosta et al., have used Western blotting to detect secreted IL-6 and IL-8 in media from senescent cells, thereby validating qRT-PCR or RNA-seq findings at the protein level [71].

Despite its strengths, Western blotting has notable limitations, since it is less sensitive than ELISA for detecting low-abundance secreted proteins and requires high-quality, validated antibodies to ensure specificity. Quantification is semi-quantitative at best, as signal intensity can be affected by factors such as antibody affinity, exposure time, and transfer efficiency. Moreover, the method is relatively low-throughput and labor-intensive, limiting its utility for large-scale screening of SASP components [78,79].

Mass Spectrometry

Mass spectrometry (MS) has emerged as a powerful and unbiased approach for the comprehensive profiling of the SASP at the protein level. Unlike targeted methods such as ELISA or Western blotting, MS-based proteomics allows for the simultaneous identification and quantification of hundreds to thousands of proteins in complex biological samples without prior assumptions. This makes it uniquely suited for uncovering the full diversity and dynamics of the SASP across different cell types, senescence triggers, and biological contexts [80]. The general workflow of mass spectrometry-based proteomics involves the extraction and digestion of proteins (commonly using trypsin) from samples such as cell culture-conditioned media, tissue interstitial fluid, or blood plasma. The resulting peptides are separated by liquid chromatography (LC) and then analyzed using tandem MS, where their mass to charge ratios are measured and sequenced to infer protein identity and abundance [43,81].

A landmark study by Basisty et al. exemplifies the application of MS in SASP research. The authors used data-independent acquisition (DIA)-based MS to systematically analyze the secretomes of multiple human cell types (fibroblasts, epithelial cells, endothelial cells, and preadipocytes) undergoing senescence induced by irradiation, oncogene activation, or replicative exhaustion. Their work produced the first comprehensive "SASP Atlas," which cataloged both core and context-specific SASP proteins—including cytokines (e.g., IL-6, CXCL1), extracellular matrix components (e.g., fibronectin, SERPINs), proteases (e.g., MMPs, cathepsins), and unexpected mediators like growth differentiation factor 15 (GDF15) and stanniocalcin 1 (STC1) [58]. These findings expanded the functional landscape of the SASP and highlighted its complexity beyond traditional inflammatory markers.

Samsonraj et al. used MS-based proteomics to analyze conditioned media from replicatively senescent bone marrow-derived human mesenchymal stem cells (MSCs), comparing them to early-passage controls. They identified secreted proteins uniquely elevated in senescent MSCs, including IL-6, MMP-1, SERPINE1, OPG, and COL1A1. These findings were validated under oxidative and irradiative stress, and select proteins (e.g., IL-6, OPG) were also increased in plasma of aged rats [44]. Özcan et al. conducted an unbiased LC–MS/MS (liquid chromatography–tandem mass spectrometry) analysis of secretomes from bone marrow and adipose mesenchymal stromal cells which were exposed to oxidative stress, irradiation, doxorubicin, and replicative exhaustion. They identified 11 proteins shared across cells subjected to senescence triggers and three key signaling pathways that could suggest a conserved core SASP signature [82].

One of the key strengths of MS in SASP research is its ability to detect post-translationally modified proteins, alternative splicing products, and proteolytically cleaved fragments that are often missed by transcriptomics or antibody-based methods [46,83]. For instance, matrix metalloproteinases (MMPs), a class of enzymes often enriched in the SASP, are secreted in latent forms and activated extracellularly—processes that are captured effectively by MS analysis of conditioned media but not by RNA-seq or standard Western blotting [47]. Moreover, MS is particularly well-suited for biomarker discovery in clinical samples [84].

Nevertheless, MS-based proteomics comes with challenges. The technique requires specialized instrumentation, expert data analysis, and careful sample preparation to minimize artifacts and improve detection sensitivity. Low-abundance cytokines and chemokines may fall below detection thresholds unless samples are fractionated or enriched. Additionally, the complexity and dynamic range of biological fluids like plasma or serum necessitate depletion of abundant proteins (e.g., albumin) to enhance SASP protein recovery [85].

Multiplex Assays (Luminex, MSD)

Multiplex immunoassays have emerged as highly valuable tools for the quantitative measurement of multiple protein analytes simultaneously. They provide distinct advantages over conventional single-analyte ELISAs for the detection of SASP components, particularly when the goal is to characterize complex secretory signatures. By enabling the simultaneous quantification of multiple cytokines, chemokines, growth factors, and proteases within a single sample, multiplex platforms allow comprehensive profiling of the diverse SASP repertoire, which typically includes IL-6, IL-8, MCP-1, matrix metalloproteinases, and insulin-like growth factor binding proteins. This parallel detection reduces the sample volume required, an important consideration when working with limited material from primary senescent cultures or small tissue biopsies. Multiplex assays also improve experimental efficiency by reducing assay time and labor compared with running multiple ELISAs. Furthermore, the ability to obtain relative abundance patterns for multiple factors from the same sample facilitates identification of core and context-specific SASP signatures. Although initial costs may be higher, multiplex approaches can offer greater overall cost-effectiveness for broad SASP profiling [86].

Two of the most widely adopted platforms for multiplexing are Luminex xMAP technology and Meso Scale Discovery (MSD) electrochemiluminescence (ECL). Both systems offer high sensitivity, broad dynamic range, and the ability to interrogate dozens of secreted factors in parallel—attributes that are particularly advantageous when profiling the SASP in tissue-derived samples, where sample quantity and composition can be limiting [87].

Luminex Assays

Luminex technology employs microsphere-based flow cytometry. Each microsphere bead is dyed with a unique fluorescent signature and coated with a capture antibody specific to a target protein. Upon incubation with a tissue lysate, the analyte binds its corresponding bead. A biotinylated detection antibody and a streptavidin-phycoerythrin conjugate are then added, and the bead-associated signal is quantified by flow-based detection. This format is particularly useful in SASP studies involving tissue homogenates, allowing researchers to simultaneously measure inflammatory cytokines (e.g., IL-6, IL-8, TNF-α), chemokines (e.g., CCL2/MCP-1, CXCL1), and matrix remodeling proteins (e.g., MMP-3, TIMPs) [45].

In early-senescent mesenchymal stromal cells (MSCs), researchers compared conditioned media from young and aged donors using an R&D Systems Luminex panel. They measured a customized SASP set (IL-1α, IL-8, IL-6, MCP-1, IL-1β, TNF-α, GRO-β, and CCL4) and also applied assays to bone marrow sera, confirming age-associated increases in these SASP cytokines [88]. Luminex assays have also been used to assess changes in SASP composition in response to therapeutic interventions. A study by Abdelgawad et al. investigated the effects of metformin on doxorubicin-induced endothelial senescence. Using Luminex assays, researchers measured the secretion levels of SASP factors such as IL-6, TNF-α, CXCL1, CXCL2, MCP-1, MCP-3, MIP-1α, and MMP-3 in the culture supernatants of senescent endothelial cells [48]. The study found that metformin treatment significantly reduced the secretion of these pro-inflammatory cytokines and chemokines, suggesting its potential in mitigating vascular inflammation associated with cellular senescence. Camell et al. used a Luminex assay to evaluate how exposure to pathogen-associated molecular pattern (PAMP) factors and treatment with senolytic drugs (e.g., fisetin, dasatinib, and quercetin) modulates the circulating SASP proteome in aged mice. The study revealed significant reductions in pro-inflammatory and pro-fibrotic SASP components, supporting the utility of proteomics in evaluating treatment efficacy and identifying biomarkers of senescent cell clearance [89].

Meso Scale Discovery (MSD) Assays

MSD technology utilizes multi-spot electrochemiluminescent detection on patterned plates, where each spot is coated with a different capture antibody. Upon sample incubation and binding of target analytes, detection antibodies labeled with electrochemiluminescent tags (e.g., ruthenium complexes) are introduced. When voltage is applied, light is emitted from each spot in proportion to analyte concentration, enabling high-sensitivity multiplex detection. MSD is particularly valued for its low background signal, broad dynamic range (up to 5–6 logs), and excellent reproducibility [90]. It has been widely used in both preclinical and clinical SASP studies.

For example, Alimbetov et al. investigated the effects of small molecule inhibitors targeting p38 MAPK and MK2 on the SASP in human fibroblasts. Conditioned media from senescent cells were analyzed using MSD multiplex immunoassays to measure cytokines such as GM-CSF, IL-1β, IL-6, IL-8, IL-10, TNF-α, and IFN-γ. The study demonstrated that these inhibitors effectively reduced the secretion of pro-inflammatory SASP factors [91]. A study by Smith et al. developed a model to examine molecular mechanisms of macrophage aging. Conditioned media from macrophages at various time points were collected and analyzed using MSD U-PLEX assays. The assays quantified levels of IL-6, KC-GRO, MCP-1, MMP-9, TNF-α, IL-1β, and IL-10, providing insights into the dynamic changes in SASP components during macrophage aging [92]. Alessio et al. studied the progression of senescence in mesenchymal stromal cells (MSCs) induced by ionizing radiation. Conditioned media from early and late senescent MSCs were analyzed using MSD assays to profile SASP components. The study revealed temporal changes in the SASP composition, highlighting differences between early and late senescent states [93].

Luminex and MSD multiplex immunoassays provide powerful, scalable platforms for the quantitative analysis of SASP components in cell culture supernatants samples. Their ability to measure dozens of analytes simultaneously from small sample volumes makes them ideal for in vivo studies of tissue aging, senescence burden, and response to senescence-targeted therapies. By enabling direct comparison of SASP expression across tissue types, conditions, and species, these assays serve as indispensable tools in both basic and translational senescence research [94]. Of course these assays have also their limitations. Cross-reactivity and varying affinity of antibodies can introduce quantification errors with validation of antibody pairs being essential. Moreover, while they allow detection of known SASP proteins, they are inherently targeted platforms and cannot detect novel or unexpected secreted factors [95]. For discovery purposes, integration with untargeted mass spectrometry remains valuable.

The detection of circulating SASP components in plasma and serum has become an increasingly important area of research, particularly in the context of aging, age-related diseases and the development of senescence-targeted therapeutics. Unlike cell culture or tissue-based studies, plasma and serum analyses provide a minimally invasive way to assess systemic senescence burden and hold potential for biomarker discovery, longitudinal monitoring, and clinical translation. Circulating SASP components are typically measured in plasma (obtained using anticoagulants like EDTA or heparin) or serum (obtained after clotting), with minor differences in protein content due to clotting-related release of platelet factors. The circulating SASP includes a diverse array of secreted proteins which are detectable in circulation, where they may contribute to systemic inflammation, tissue dysfunction, or inter-organ communication during aging and disease [29,96].

Detection Methods

A variety of techniques are used to measure SASP components in plasma and serum. Multiplex immunoassays (Luminex, MSD) offer high-throughput measurement of dozens of proteins. Picca et al. utilized the Bio-Plex Pro Human Cytokine 27-plex Assay (Luminex xMAP technology) and the ELLA™ automated immunoassay system to identify elevated levels of inflammatory and senescence-related markers (IL-1β, IL-6, TNF-α, activin A, FGF21, GDF15, GFAP, ICAM-1, SERPINE1, and TIMP-1) in older adults with physical frailty and sarcopenia, suggesting a link between circulating SASP factors and age-related physical decline [97]. Results from LIFE Study concerning SASP markers and mild cognitive impairment found associations between higher plasma levels of certain SASP markers and increased odds of mild cognitive impairment and dementia over a 24-month period. Using Luminex xMAP multiplex magnetic bead-based immunoassays analyzed on the MAGPIX System, they assessed 27 SASP biomarkers, including MPO, MMP-1, and MMP-7 [98]. Manna et al. demonstrated heterogeneous differences in SASP profiles in maternal plasma samples from cases of pre-eclampsia and intrauterine growth restriction (IUGR), indicating potential biomarkers for these conditions measuring IL-6, IL-8, IL-13, IL-1α, IFN-γ, MCP-1, MIP-1α, and MMP-3. They utilized the Human U-Plex kit and MMP-3 Ultra-sensitive kit from MSD [49].

ELISA is highly sensitive for individual factors. In many studies, it was combined with multiplex immunoassays to measure specific SASP components. In a study by Schafer et al., ELISAs were used to quantify plasma levels of SASP proteins activin A and plasminogen activator inhibitor 2 (PAI-2) in order to find a correlation between their levels and chronological age, frailty, and age-related pathologies [29]. Wang et al. measured the plasma levels of circulating GDF15 as potential biomarker of metabolic dysregulation and aging in people living with HIV [99].

The ability to detect SASP factors in plasma and serum provides a foundation for the development of senescence biomarkers that could be used in aging and frailty assessments, prediction of chronic disease risk (e.g., cardiovascular disease, osteoarthritis, neurodegeneration), monitoring of senescence-modifying interventions (e.g., senolytics, caloric restriction, exercise), and stratification of patients for clinical trials [93, 94]. The analysis of circulating SASP components in plasma and serum provides a crucial bridge between experimental senescence research and clinical application [51]. The integration of machine learning and systems biology approaches with large-scale proteomic and clinical datasets is expected to further refine SASP signatures and improve their specificity for senescence [50]. Additionally, longitudinal studies tracking SASP factors in plasma/serum across lifespan or in response to therapies will be key for establishing robust, clinically meaningful biomarkers.

Despite its promise, the detection of circulating SASP components poses several challenges. SASP factors secreted by senescent cells may be diluted in the bloodstream, making them harder to detect, particularly for proteins with short half-lives. Circulating proteins may arise from multiple cell types, not exclusively senescent cells, necessitating complementary methods to confirm senescence origin. Moreover, many SASP proteins overlap with general inflammatory markers, complicating the interpretation in settings of infection, injury, or other comorbidities [100,101]. Lastly, collection, processing, and storage of plasma/serum samples can influence protein integrity and reproducibility.

Protein Localization

Understanding the localization of SASP proteins is critical for elucidating their functional roles, cellular origins, and tissue-level impacts in the context of cellular senescence. While bulk measurements (e.g., ELISA, mass spectrometry) can quantify the abundance of SASP components in culture media, tissue lysates, or circulation, they often lack spatial resolution. Protein localization techniques enable researchers to determine where within tissues or cells SASP factors are produced, stored, or act, providing crucial insights into senescence biology and its systemic effects. SASP molecules function not only in autocrine or paracrine signaling but also diffuse into surrounding tissues and circulation [102]. Therefore, establishing precise spatial relationships, such as whether SASP proteins are localized to senescent cells themselves, neighboring immune cells, or extracellular compartments, is fundamental to understanding their mechanisms of action [103]. Several well-established and emerging techniques are employed to visualize and localize SASP proteins in situ, typically within tissues or cell populations [104].

(i) Immunohistochemistry

Immunohistochemistry (IHC) uses antibody-based detection of proteins in formalin-fixed, paraffin-embedded (FFPE) or frozen tissue sections. A chromogenic or fluorescent label is used to visualize the antibody–protein complex. Studies demonstrate the utility of IHC in localizing and quantifying SASP components within human tissues, providing insights into the role of cellular senescence in various pathological conditions [105,106]. Vital et al. assessed the involvement of SASP factors in benign prostatic hyperplasia (BPH), using IHC analysis to detect the expression of cathepsin D and IL-1α in BPH tissues. Multifocal epithelial expression of both were observed, suggesting a role for SASP in the development of BPH [107].

(ii) Immunofluorescence

Immunofluorescence (IF) is a variant of IHC using fluorescently labeled antibodies. Fluorescently labeled antibodies bind to target proteins, and when excited by specific wavelengths of light, these fluorophores emit fluorescence that can be detected using fluorescence microscopy [108]. It is commonly used in both tissue sections and cultured cells. Xu et al. created a reversible immortalized cell line by expressing SV40T in human keratinocytes. Along with other methods they used IF to detect SASP-associated proteins MMP-1, SERPINB2, and VEGF-A after inducing a senescent phenotype by terminating SV40T expression [109]. Ruhland et al. used immunofluorescence staining to detect increased expression of IL-6 in stromal fibroblasts of mouse skin grafts when senescence was present [110].