sa β gal staining solution  (Cell Signaling Technology Inc)

Journal: Biochemistry and Biophysics Reports

Article Title: Screening of metabolic-related biomarkers linking intervertebral disc degeneration and type 2 diabetes based on comprehensive bioinformatics analysis and machine learning

doi: 10.1016/j.bbrep.2026.102593

Figure Lengend Snippet: BCAA metabolism and immune changes in degenerated NP cells. (A) qRT-PCR was conducted to analysis the mRNA levels of BCAA metabolism-related enzymes and BCAA transporter in grade I/II and grade III/IV NP tissues; n = 20; P < 0.05. (B, C) Expression levels of mRNA for pro-inflammatory factors, BCAA metabolism-related enzymes, and BCAA transporters in control group and TNF-α-treated HNPC cells. (D) Expression of SA-β-gal in HNPC cells stimulated with different concentrations of TNF-α. (E, F) p16, p21, p53 mRNA level in TNF-α-stimulated HNPC cells,and correlation analysis with TNF-α concentration.

Article Snippet: Cellular senescence was assessed using Senescence-Associated β-Galactosidase (SA-β-gal) staining (Senescence β-Galactosidase Staining Kit, #C0602, Beyotime, China), performed according to the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Concentration Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Comparative analysis of SCS and D + Q drugs on glucocorticoid-induced bone marrow senescence inhibition. ( A ) Schematic diagram showing the treatment of SCS and D + Q after glucocorticoid-induced senescence. ( B and C ) Representative flow cytometry images of bone marrow SA-β-Gal for senescence detection on day 42 (B), with corresponding quantification analysis (C). n = 6 biological replicates. ( D and E ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( F ) Schematic diagram of SCS and D + Q treatment in the early stage of glucocorticoid-induced senescence. ( G and H ) Representative flow cytometry images of p16-positive senescent cells in bone marrow on day 42 (G), with corresponding quantification analysis (H). n = 6 biological replicates. ( I and J ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( K-M ) Representative images of HE staining of the distal femur with macro and high-magnification images (K), and quantification of trabecular and cortical bone empty lacunae (L and M). n = 6 biological replicates. (Scale bars, 550 μm and 25 μm) ( N and O ) Representative ALP staining images of in vitro osteogenic differentiation of bone marrow LepR + MSCs after 14 days (N), with corresponding quantification analysis (O). n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C , D , E , H , I , J , L , M and O ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Inhibition, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Journal of Pharmaceutical Analysis

Article Title: Novel bioactive peptides targeting Keap1-Nrf2 interaction for combating UVA-induced skin aging: Computational discovery and experimental validation

doi: 10.1016/j.jpha.2025.101446

Figure Lengend Snippet: Protective effects of Seq1 and Seq3 on ultraviolet A (UVA)-induced cellular aging in human keratinocytes (HaCaT) cells. (A) The effect of peptide treatment on the migration of HaCaT cells. (B) The extent of wound closure was quantified and depicted on a histogram. (C) Assessment of the effects of peptides on senescence associated β-galactosidase (SA-β-Gal) activity in HaCaT cells. (D) Quantitation of SA-β-gal positive cells in HaCaT cells. (E) Immunofluorescence (IF) analysis of phosphorylated γ-H2AX in response to UVA irradiation and peptide treatment at varying concentrations to determine their influence. (F) 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining reveals intracellular reactive oxygen species (ROS) levels in UVA-irradiated HaCaT cells following peptide or tert-butylhydroquinone (t-BHQ) treatment. (G) Fluorescence intensity of ROS. (H) Western blot analysis of Seq1 and Seq3 on the expression of matrix metalloproteinase (MMP)-1 and MMP-9 in UVA-induced HaCat cells. (I) Relative MMP-1 and MMP-9 messenger RNA (mRNA) levels in Seq1 and Seq3 treated UVA-induced HaCat cells. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). (J) Western blot analysis showing the change of inducible nitric oxide synthase (iNOS) and interleukin-1 beta (IL-1β) in HaCat cells. (K) Quantitation of IL-1β and tumor necrosis factor-alpha ( TNF-α ) released by HaCat cells by quantitative real-time polymerase chain reaction (qRT-PCR). (L) Glutathione peroxidase (GSH-Px) activity levels in HaCaT cells. (M) Measurement of superoxide dismutase (SOD) activity levels in HaCaT cells. (N) Nrf2-dependent antioxidant enzymes protein levels for reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase modifier subunit (GCLM) in UVA-irradiated HaCaT cells treated with peptides. Protein expression normalized to GAPDH. (O) Quantitation of GCLM , HO-1 , and NQO1 released by HaCat cells by qRT-PCR. Unless otherwise indicated in the figure, the peptide concentration was 20 μM. All data are presented as means ± standard deviation (SD) ( n = 3). Statistical significance is denoted by ∗ P < 0.05 , ∗∗ P < 0.01 , ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 vs. UVA-irradiated group.

Article Snippet: To evaluate cellular senescence induced by peptides and UVA treatments, HaCaT cells were stained using the SA-β-Gal Staining Kit (C0602, Beyotime, Shanghai, China), following the manufacturer's guidelines.

Techniques: Migration, Activity Assay, Quantitation Assay, Immunofluorescence, Irradiation, Staining, Fluorescence, Western Blot, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Standard Deviation

Journal: Antioxidants

Article Title: 2-Methoxystypandrone from Polygonum cuspidatum Rejuvenates Senescence by Reducing Mitochondrial ROS

doi: 10.3390/antiox15030357

Figure Lengend Snippet: 2-MS improves senescence-associated phenotypes. Senescent fibroblasts treated with DMSO (0.01%) or 2-MS (4, 8, 12 μM) at 4-day intervals for 12 days. Then, autofluorescence ( A ), senescence-associated β-galactosidase (SA-β-gal) ( B ), p16 ( C ), cellular proliferation ( D ), CXCL12 ( E ), SLIT2 ( F ), COL1A2 ( G ), MMP-1 ( H ), and HYAL1 expression ( I ) were measured. ( A ) Representative flow graphs of autofluorescence. ( B ) + indicates SA-β galactosidase-positive cells. − indicates SA-β galactosidase negative cells. Scale bar: 10 μm. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test, with results considered not significant (n.s.) or significant at ** p < 0.01. Data represent the mean ± S.D., n = 3.

Article Snippet: For SA-β-Gal staining (9860; Cell Signaling Technology, Beverly, MA, USA), the manufacturer’s instructions were adhered to.

Techniques: Expressing