tunel based titertacs colorimetric apoptosis detection kit  (R&D Systems)

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: GF conditions attenuate Cer-induced AP and systemic inflammation. (A) Schematic overview of the experimental design. Male C57BL/6 mice (7–8 weeks old) housed under SPF or GF conditions were administered hourly intraperitoneal injections of cerulein (100 μg/kg) for 10 hours. Mice were euthanized 24 hours after the final injection for subsequent analyzes (n = 6). (B) Serum levels of pancreatic enzymes (amylase and lipase) were significantly lower in GF_Cer mice than in SPF_Cer mice, indicating attenuated pancreatic injury. (C) Representative H&E staining of pancreatic tissue (scale bar: H&E, 25 μm; TUNEL, 50 μm) revealed histopathological differences between groups, with semi-quantitative scoring of edema, inflammatory infiltration, and acinar necrosis. Apoptosis was assessed by TUNEL staining with quantification. (D) Immunohistochemical analyzes of pancreatic tissue revealed reduced infiltration of MPO⁺ neutrophils and F4/80⁺ macrophages (scale bars: 25 μm). (E) Markers of intestinal barrier dysfunction, including serum LPS and D-lactate, were also significantly reduced under GF conditions. (F) Circulating levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) were markedly decreased in GF mice, reflecting suppression of systemic inflammatory responses. Data are presented as mean ± SEM. Statistical comparisons were made using an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: GF, germ-free; SPF, specific pathogen-free; Cer, cerulein; AP, acute pancreatitis; H&E, hematoxylin and eosin; MPO, myeloperoxidase; LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

Article Snippet: Apoptotic cells in pancreatic tissue sections were detected using a fluorescein-based TUNEL apoptosis detection kit (Servicebio, China), following the manufacturer's instructions.

Techniques: Injection, Staining, TUNEL Assay, Immunohistochemical staining, Two Tailed Test

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: FMT from AP patients exacerbates pancreatic injury and NETs formation in GF mice. (A) Schematic representation of experimental design: GF mice received FMT from either healthy controls (FMT-HC) or acute pancreatitis (FMT-AP) patients for two weeks. AP was induced by 10 hourly intraperitoneal injections of Cer (100 μg/kg). Mice were sacrificed 24 hours after the initial injection. (B) Representative H&E staining of pancreatic tissues (scale bar, 25 μm), with histological scores of edema, inflammatory infiltration, and necrosis. (C) Immunohistochemical analysis and quantification of pancreatic infiltration by neutrophils (MPO), macrophages (F4/80), and apoptotic cell death (TUNEL assay) (scale bars: MPO, F4/80, 25 μm; TUNEL, 50 μm). (D) Flow cytometric analysis and quantification of CD11b + Ly6G + neutrophils (gated on CD45 + cells) in spleen and bone marrow. (E) Immunofluorescence staining and quantification of NETs formation in pancreatic tissue, defined by co-expression of citrullinated histone H3 (CitH3) and MPO (CitH3 + MPO + cells; scale bar, 50 μm). (F) Serum cfDNA concentrations, serving as an indicator of systemic NETs release. (G) Serum levels of amylase and lipase, indicative of pancreatic injury severity. (H) Serum levels of pro-inflammatory cytokine (TNF-α, IL-6, IL-1β) and LPS, reflecting systemic inflammation and intestinal barrier disruption. (I) qRT-PCR analysis of pancreatic tissue for pro-inflammatory cytokines (Il6, Tnfa, Il1β ), neutrophil chemotaxis and activation markers ( Cxcl1, Cxcl2, S100a8, S100a9 ), and NETs-related genes ( Padi4, Nfkb, Nlrp3, Il17a ). Data are presented as mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t-test (* p <0.05, ** p <0.01, *** p <0.001). Abbreviations: FMT, fecal microbiota transplantation; AP, acute pancreatitis; GF, germ-free; Cer, cerulein; HC, healthy control; MPO, myeloperoxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CitH3, citrullinated histone H3; cfDNA, cell-free DNA; LPS, lipopolysaccharide; NETs, neutrophil extracellular traps; qRT-PCR, real-time quantitative PCR.

Article Snippet: Apoptotic cells in pancreatic tissue sections were detected using a fluorescein-based TUNEL apoptosis detection kit (Servicebio, China), following the manufacturer's instructions.

Techniques: Injection, Staining, Immunohistochemical staining, TUNEL Assay, Immunofluorescence, Expressing, Disruption, Quantitative RT-PCR, Chemotaxis Assay, Activation Assay, Two Tailed Test, Transplantation Assay, Control, Real-time Polymerase Chain Reaction

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: Selective depletion of Gram-negative bacteria attenuates cerulein-induced pancreatic inflammation and NETs formation, whereas depletion of Gram-positive bacteria fails to confer protection. (A) Schematic overview of the experimental design: SPF mice were pretreated orally with PBS, neomycin (NEO; targeting Gram-negative bacteria), or vancomycin (VAN; targeting Gram-positive bacteria) for one week, followed by hourly intraperitoneal injections of cerulein (Cer, 100 μg/kg, 10 doses). Mice were sacrificed 24 h after the initial injection. (B) Representative H&E staining of pancreatic tissue (scale bar, 25 μm) with corresponding histological scoring for edema, inflammatory infiltration, necrosis. (C) Immunohistochemical staining and quantification of pancreatic neutrophils (MPO), macrophages (F4/80), and apoptotic cells (TUNEL assay) (scale bars: MPO and F4/80, 25 μm; TUNEL, 50 μm). (D) Flow cytometric analysis and quantification of neutrophils (CD11b + Ly6G + , gated on CD45 + cells) in spleen and bone marrow. (E) qRT-PCR analysis of pancreatic expression levels of pro-inflammatory cytokines ( Il6, Tnfa, Il1β ), neutrophil chemotaxis and activation markers ( Cxcl1, Cxcl2, S100a8, S100a9 ), and genes involved in NETosis ( Padi4, Nfkb, Nlrp3, Il17a ). (F) Immunofluorescence staining and quantification of NETs formation (MPO + CitH3 + cells) in pancreatic tissues (scale bar, 50 μm). (G) Serum concentrations of cfDNA assessed by ELISA. (H–I) Serum levels of pancreatic enzymes (amylase and lipase) and systemic inflammatory markers (TNF-α, IL-6, IL-1β), as well as lipopolysaccharide (LPS), indicating intestinal barrier disruption. Data are presented as mean ± SEM. Statistical significance was determined by an unpaired two-tailed Student’s t-test (* p <0.05, ** p <0.01, *** p <0.001; ns, not significant). Abbreviations: AP, acute pancreatitis; SPF, specific pathogen-free; Cer, cerulein; NEO, neomycin; VAN, vancomycin; MPO, myeloperoxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; CitH3, citrullinated histone H3; cfDNA, cell-free DNA; NETs, neutrophil extracellular trap; qRT-PCR, real-time quantitative PCR; LPS, lipopolysaccharide.

Article Snippet: Apoptotic cells in pancreatic tissue sections were detected using a fluorescein-based TUNEL apoptosis detection kit (Servicebio, China), following the manufacturer's instructions.

Techniques: Bacteria, Injection, Staining, Immunohistochemical staining, TUNEL Assay, Quantitative RT-PCR, Expressing, Chemotaxis Assay, Activation Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Disruption, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: E. coli exacerbates cerulein-induced AP and promotes NETs formation in GF mice. (A) Schematic overview of the experimental protocol: GF mice were orally administered PBS or a pathogenic strain of E. coli (ATCC 43895) for one week, followed by hourly intraperitoneal injections of cerulein (100 μg/kg, 10 doses in total). Mice were sacrificed 24 h after the initial injection. (B) Representative H&E staining of pancreatic tissues (scale bar, 25 μm), with corresponding histological scoring for edema, inflammatory infiltration, and necrosis. (C) Immunohistochemical staining and quantification of neutrophils (MPO), macrophages (F4/80), and apoptotic cells (TUNEL assay) in pancreatic tissue sections (scale bars: MPO and F4/80, 25 μm; TUNEL, 50 μm). (D) Serum amylase and lipase levels as biochemical indicators of pancreatic injury. (E) Serum concentrations of inflammatory cytokines (TNF-α, IL-6, IL-1β) and LPS, reflecting systemic inflammatory responses and gut barrier disruption. (F) Flow cytometric analysis and quantification of CD11b + Ly6G + neutrophils (gated on CD45 + cells) in spleen and bone marrow. (G) Immunofluorescence staining and quantification of pancreatic tissue showing NETs formation (CitH3 + MPO + cells) (scale bar, 50 μm). (H) Serum levels of cfDNA measured by ELISA. (I) qRT-PCR analysis of pancreatic tissue expression of pro-inflammatory cytokines ( Il6, Tnfa, Il1β ), neutrophil chemotaxis and activation markers ( Cxcl1, Cxcl2, S100a8, S100a9 ), and genes associated with NETs formation ( Padi4, Nfkb, Nlrp3, Il17a ). Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t-test. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: GF, germ-free; Cer, cerulein; AP, acute pancreatitis; MPO, myeloperoxidase; CitH3, citrullinated histone H3; cfDNA, cell-free DNA; LPS, lipopolysaccharide; NETs, neutrophil extracellular traps; qRT-PCR, real-time quantitative PCR.

Article Snippet: Apoptotic cells in pancreatic tissue sections were detected using a fluorescein-based TUNEL apoptosis detection kit (Servicebio, China), following the manufacturer's instructions.

Techniques: Injection, Staining, Immunohistochemical staining, TUNEL Assay, Disruption, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Chemotaxis Assay, Activation Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Gut Microbes

Article Title: Gut microbiota dysbiosis exacerbates acute pancreatitis via Escherichia coli -driven neutrophil heterogeneity and NETosis

doi: 10.1080/19490976.2025.2606480

Figure Lengend Snippet: Single-cell transcriptomic analysis reveals gut microbiota dysbiosis promotes NETs formation via neutrophil Subset Reprogramming. (A) UMAP visualization of pancreatic single-cell RNA sequencing data identifies five transcriptionally distinct neutrophil clusters (Neutrophils_1 to Neutrophils_5). (B) UMAP plot illustrating differences in the distribution of neutrophil subsets between SPF_Cer and GF_Cer mice, demonstrating microbiota-dependent alterations in neutrophil heterogeneity. (C) Bar plot quantifying the relative abundance of neutrophil subpopulations across SPF_Cer and GF_Cer groups, highlighting the expansion of specific subsets in dysbiotic conditions. (D) Heatmap illustrating the expression profiles of representative genes across neutrophil subsets, delineating their functional identities. (E) KEGG pathway enrichment analysis of neutrophil clusters reveals distinct molecular signatures: Neutrophils_2 enriched in pro-inflammatory pathways including TNF, NF-κB, IL-17 signaling, apoptosis, and necroptosis; Neutrophils_3 enriched in antigen processing and Toll-like receptor signaling; Neutrophils_4 characterized by enrichment in HIF-1 signaling, glycolysis, lysosome activity, and autophagy- associated metabolic pathways. (F) Dot plot profiling key genes associated with anti-inflammatory regulation ( Hmox1 and Il1rn ), mitochondrial protection ( Bnip3 and Nupr1 ), metabolic reprogramming ( Ldha and Aldoa ), and tissue repair ( Reg1 and Reg2 ) across neutrophil subpopulations. (G) GSEA focused on the Neutrophils_4 demonstrates significant enrichment of NETs formation pathways in SPF_Cer mice compared to GF_Cer mice, suggesting involvement of this subset in NETs regulation under dysbiotic conditions. Abbreviations: UMAP, uniform manifold approximation and projection; GSEA, gene set enrichment analysis; NETs, neutrophil extracellular traps; SPF, specific pathogen-free; GF, germ-free; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Apoptotic cells in pancreatic tissue sections were detected using a fluorescein-based TUNEL apoptosis detection kit (Servicebio, China), following the manufacturer's instructions.

Techniques: RNA Sequencing, Expressing, Functional Assay, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

doi: 10.1172/jci171063

Figure Lengend Snippet: Figure 4. CoREST inhibition reduces colony formation and induces apoptosis in BRAFi-R melanoma cells with or without BRAFi. (A) Colony formation assay quantification in 451Lu-R (left) and 1205Lu-R (right) melanoma cells treated with DMSO, 5 μM PLX4032 alone, 2.5 μM corin alone, or 2.5 μM corin plus 5 μM PLX4032 for 10 days (n = 3). (B) Quantification of TUNEL+ cells in 451Lu-R and 1205Lu-R melanoma cells following 72 hours of treatment with DMSO, 5 μM PLX4032 alone, 1 μM corin alone, or 1 μM corin plus 5 μM PLX4032 (n = 3). (C) Senescence-associated β-galactosidase staining of 451Lu-R melanoma cells treated with DMSO, 5 μM PLX4032 alone, 1 μM corin alone, or 1 μM corin plus 5 μM PLX4032 for 72 hours and quantification (n = 2–3). Scale bar: 50 μm. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 1-way ANOVA with Tukey’s test.

Article Snippet: Apoptosis was detected used the TUNEL-based TiterTACS Colorimetric Apoptosis Detection Kit (R&D Systems).

Techniques: Inhibition, Colony Assay, TUNEL Assay, Staining

Journal: BMC Cardiovascular Disorders

Article Title: Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification

doi: 10.1186/1471-2261-14-29

Figure Lengend Snippet: After 6 days of in vitro culture, VICs in collagen-deficient leaflets exhibited significantly higher levels of both proliferation and apoptosis. (A) Proliferating cells per total cell number on Day 6, and (B) Apoptotic cells per total cell number on Day 6, expressed as percent of no-enzyme control. *P <0.0005 compared to undepleted.

Article Snippet: Apoptotic VICs in the leaflets were visualized using the TUNEL-based ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA).

Techniques: In Vitro

Journal: Breast Cancer Research : BCR

Article Title: Scavenger receptor class B type I regulates cellular cholesterol metabolism and cell signaling associated with breast cancer development

doi: 10.1186/bcr3483

Figure Lengend Snippet: Knockdown of SR-BI inhibits xenograft tumor growth in vivo. (A) Knockdown of SR-BI results in decreased angiogenesis in MDA-MB-231 xenograft tumors. Immunohistochemistry was performed to determine expression of CD31. CD31 staining was quantified by averaging the number of vessels per field in five separate fields per sample. Columns represent the mean microvessel density; bars represent ± SD. Microvessel density was statistically greater in shCTL MDA-MB-231 tumors compared with shSRBI MDA-MB-231 cells (* P < 0.05 Student's t test; n = 4 per group). (B) Knockdown of SR-BI results in increased apoptosis in MDA-MB-231 xenograft tumors. Images shown are representative of TUNEL assay staining. Percentage of TUNEL-positive cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells in four separate fields. Columns represent the percentage of TUNEL-positive cells; bars represent ± SD. TUNEL staining is statistically increased in shSRBI MDA-MB-231 tumors compared with shCTL MDA-MB-231 tumors (*** P < 0.001, Student's t test, shCTL n = 3, and shSRBI n = 4).

Article Snippet: Apoptosis was measured with TUNEL assay by using the TUNEL-based ApopTag Peroxidase In Situ Apoptosis Detection Kit from Millipore (Temecula, CA, USA), as per manufacturer’s instructions.

Techniques: In Vivo, Immunohistochemistry, Expressing, Staining, TUNEL Assay