elisa kits for mcp1  (R&D Systems)

Journal: Redox Biology

Article Title: The electrophilic metabolite of kynurenine, kynurenine-CKA, requires C151 in Keap1 to derepress Nrf2

doi: 10.1016/j.redox.2026.104009

Figure Lengend Snippet: AhR inhibition has no effect on the anti-inflammatory actions of Kyn-CKA in BMDMs. (A) Mouse AhR reporter cells expressing luciferase under the control of the xenobiotic response element (XRE) were incubated with Kyn-CKA or kynurenine for 24 h prior to luminescence measurement (n = 3). Data were fitted to a sigmoidal four-parameter logistic curve (L-Kyn: r 2 = 0.824, IC 50 = 28 μM, span = 3218 RFU. Kyn-CKA: r 2 = 0.970, IC 50 = 13 μM, span = 47,901 RFU). (B) Inhibition of AhR-dependent luciferase expression by CH223191 (AhRinh). (C–F) Kyn-CKA inhibits the expression of the NF-κB-regulated genes IL6, MCP1, Nos2 and IL1β in BMDM following 5 h incubation with LPS (0.5 μg/mL) and the indicated concentrations of Kyn-CKA both in the presence or absence of CH223191 (10 μM). Data are n = 3–4, ∗∗∗∗p < 0.0001 by one-way ANOVA and Tukey's post-test. (G – H) Extracellular cytokine levels from BMDM treated as in C–F for 5 h. Data are n = 3–4 (all replicates shown), ∗∗∗∗p < 0.0001 by one-way ANOVA and Tukey's post-test.

Article Snippet: ELISA kits for MCP1 (DY479 and MJE00B) and IL6 (DY406) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Expressing, Luciferase, Control, Incubation

Journal: Redox Biology

Article Title: The electrophilic metabolite of kynurenine, kynurenine-CKA, requires C151 in Keap1 to derepress Nrf2

doi: 10.1016/j.redox.2026.104009

Figure Lengend Snippet: The low-dose anti-inflammatory effects of Kyn-CKA in BMDMs are dependent on Nrf2. (A – B) Treatment with Kyn-CKA (5 h) fails to induce the expression of Nqo1 and Gclm in BMDMs obtained from Nrf2-knockout (Nrf2 −/− ) mice. Data are n = 3, ∗∗∗∗p < 0.0001 by two-way ANOVA and Tukey's post-test. (C – G) Effects of Kyn-CKA on the expression of NF-κB−regulated genes in WT and Nrf2-KO BMDMs following 5 h incubation with LPS (0.5 μg/mL) and the indicated concentrations of Kyn-CKA. Data are n = 6, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA and Tukey's post-test. Nrf2 was identified as a significant source of variation for MCP1, IL6, TNFα, Nos2, (p < 0.0001) and IL1β (p < 0.005).

Article Snippet: ELISA kits for MCP1 (DY479 and MJE00B) and IL6 (DY406) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Knock-Out, Incubation

Journal: Metabolism: clinical and experimental

Article Title: The serine protease KLK7 promotes immune cell infiltration in visceral adipose tissue in obesity.

doi: 10.1016/j.metabol.2025.156239

Figure Lengend Snippet: Fig. 1. Klk7 expression in immune cells is significantly altered in obesity. a) RT-PCR analysis reveals higher expression of Klk7 in bone marrow derived-macrophages (BMDM) isolated from HFD-fed (filled blue) when compared to chow-fed (outlined blue) wild type (WT) mice (n = 4). mRNA levels are relative to BMDM from chow- fed mice. b) Experimental set-up to assess the effect of conditioned media (CM) obtained from eWAT explants of HFD-fed WT mice, in cytokine expression (c) and KLK7 release (d) from BMDM isolated from chow-fed WT mice, after a period of six hours. c) RT-PCR analysis reveals significant induction of gene expression of Tnfa and Mcp1 in CM-treated (red) BMDM compared to DMEM controls (black) (n = 3 per condition). mRNA levels are relative to controls. d) ELISA measurement reveals the presence of mouse KLK7 in CM (red -, n = 3) and further significant release of KLK7 from CM treated BMDM (red +, n = 5), compared to controls (DMEM, black, n = 5). Treatment was for 6 h without serum. e) RT-PCR analysis reveals higher Mcp1 expression in BMDM from chow-fed WT mice treated with recombinant KLK7 over 24 h (turquoise, n = 9), compared to controls (black, n = 8). Significance (p < 0.05) was determined by unpaired two-tailed t-tests (a, e), multiple corrected unpaired two-tailed t-test (c) or by one-way ANOVA and Tukey's post-tests (d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We used ELISA to analyze serum levels of insulin (Mouse Insulin ELISA, Mercodia, Uppsala, Sweden), C-peptide (Mouse C-peptide ELISA, ALPCO, Salem, USA), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, USA), adiponectin (Mouse Adiponectin ELISA, AdipoGen, San Diego, USA), MCP1 (Mouse/rat CCL2/JE/MCP-1 Quantikine ELISA, R&D Systems, Minneapolis, USA), TNFA (#43904, BioLegend, San Diego, USA) and KLK7 (E03K0074, Bluegene, Shanghai, China) according to the manufacturer's protocols.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Gene Expression, Enzyme-linked Immunosorbent Assay, Recombinant, Two Tailed Test

Journal: Metabolism: clinical and experimental

Article Title: The serine protease KLK7 promotes immune cell infiltration in visceral adipose tissue in obesity.

doi: 10.1016/j.metabol.2025.156239

Figure Lengend Snippet: Fig. 3. Klk7 ablation in macrophages ameliorates metabolic consequences of diet-induced obesity. a) Fasted glucose levels in KLK7MKO mice and WT littermates under chow and HFD conditions (n = 15–28 per genotype) measured at the age of 22 weeks. b-e) Intraperitoneal glucose (b) and insulin (c) tolerance tests (GTT at 22 weeks and ITT at 24 weeks of age), areas under/over the curve (d) and ITT effect (5 decrease of blood glucose levels after 60 min (e)) reveals higher insulin sensitivity in HFD-fed KLK7MKO mice compared to WT littermates (n = 6–8 per genotype). f-n) ELISA measurements in serum of KLK7MKO mice and WT littermates after 20 weeks on a HFD: (f) KLK7 levels (n = 5 per genotype), (g) TNFa, and (h) MCP1 (n = 8–19 per genotype); (i) leptin, (j) adiponectin and (k) resulting leptin/adiponectin ratio (n = 9–10 per genotype); (l) insulin, (m) C-peptide and (n) resulting insulin/c-peptide ratio (n = 13–14 per genotype) establish an overall improved metabolic phenotype of HFD-fed KLK7MKO mice compared to WT littermates. Genotypes are illustrated in blue (WT) and green (KLK7MKO), diets by outlined (chow) or filled (HFD) symbols. Significance (p < 0.05) was determined by one-way ANOVA and Tukey's post-test (a), two-way ANOVA with Sidak's post-test (b-c) or unpaired two- tailed t-tests (d-n). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We used ELISA to analyze serum levels of insulin (Mouse Insulin ELISA, Mercodia, Uppsala, Sweden), C-peptide (Mouse C-peptide ELISA, ALPCO, Salem, USA), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, USA), adiponectin (Mouse Adiponectin ELISA, AdipoGen, San Diego, USA), MCP1 (Mouse/rat CCL2/JE/MCP-1 Quantikine ELISA, R&D Systems, Minneapolis, USA), TNFA (#43904, BioLegend, San Diego, USA) and KLK7 (E03K0074, Bluegene, Shanghai, China) according to the manufacturer's protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Metabolism: clinical and experimental

Article Title: The serine protease KLK7 promotes immune cell infiltration in visceral adipose tissue in obesity.

doi: 10.1016/j.metabol.2025.156239

Figure Lengend Snippet: Fig. 4. Klk7 ablation in macrophages alters adipose tissue macrophage infiltration and polarization. a) ELISA measurement reveals lower MCP1 released from naïve M0 BMDM from HFD-fed KLK7MKO mice when compared to WT littermates (n = 5 per genotype). b) ELISA measurements in epidydimal (eWAT) and inguinal (iWAT) adipose tissue lysates reveal lower MCP1 levels specifically in the eWAT from HFD-fed KLK7MKO mice when compared to WT littermates (n = 7–8 per genotype). c) Normalized pro-inflammatory cytokines abundance significantly differentially expressed across adipose tissue lysates (eWAT and iWAT) and measured via the Olink Target 48 panel unravel a fat-pad specific reduction in HFD-fed KLK7MKO mice when compared WT littermates (n = 7–8 per genotype). d) Repre sentative CD68 staining reveals a reduction in the presence of infiltrated macrophages in the eWAT from HFD-fed KLK7MKO mice when compared to WT littermates (20× magnification, scale = 100μm). e-f) Analysis of the adipocyte size (expressed as area) distribution determined from H&E stained slides reveals a higher number of smaller and a lower number of bigger adipocytes in eWAT and iWAT from HFD-fed KLK7MKO mice when compared to WT littermates (n = 5 per genotype). g) Corresponding quantification from (d) reveals a reduction in the CD68+ area of eWAT stained slides from HFD-fed KLK7MKO mice when compared to WT littermates (n = 6 per genotype). h) Quantification of ATM (enriched in F4/80) isolated from eWAT reveals a reduction in HFD-fed KLK7MKO mice when compared to WT littermates (n = 4–5 per genotype). i-j) Quantification of fluorescence activated cell sorting of eWAT-ATM with different polarization states (M1, M2 and Mme) determined through specific cell-surface markers (CD11c, CD206 and LAMP-1/CD36 respectively) reveals a higher percentage of M2 polarized ATM in the eWAT from HFD-fed KLK7MKO mice when compared to WT littermates (n = 4 per genotype) (i); and corresponding M1/M2 ratio (j). k-l) RT-PCR analysis of pro- (Tnfa and Il1b) and anti- (Retnla) inflammatory marker genes in eWAT (k) and iWAT ATM (l) reveals lower levels of inflammation in HFD-fed KLK7MKO mice when compared to WT littermates (n = 6 per genotype). mRNA levels are relative to WT littermates. m) RT-PCR analysis of Retnla expression in liver, eWAT and iWAT tissue from HFD-fed KLK7MKO mice and WT littermates (n = 5–6 per genotype). mRNA levels are relative to WT littermates (WAT to eWAT). n) RT-PCR analysis in ATM (from eWAT and iWAT) from chow-fed WT littermates treated with recombinant KLK7 over 24 h (n = 5–6 per group) reveals a fat-pad specific induction of Mcp1 in eWAT- ATM. mRNA levels are relative to eWAT controls. o) RT-PCR analysis in eWAT-ATM from chow-fed WT mice polarized to M1 while treated with recombinant KLK7 over 24 h (n = 4–5 per group) reveals a lower Retnla expression in the treated group. Genotypes and treatments are illustrated in blue (WT) and green (KLK7MKO), black (control) and turquois (KLK7). Significance (p < 0.05) was determined by unpaired two-tailed t-test (a,g,h,j,o), multiple unpaired uncorrected two-tailed t-tests (e,f,k-m), two-way ANOVA with Tukey's (b), or uncorrected Fischer's LSD (i). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We used ELISA to analyze serum levels of insulin (Mouse Insulin ELISA, Mercodia, Uppsala, Sweden), C-peptide (Mouse C-peptide ELISA, ALPCO, Salem, USA), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, USA), adiponectin (Mouse Adiponectin ELISA, AdipoGen, San Diego, USA), MCP1 (Mouse/rat CCL2/JE/MCP-1 Quantikine ELISA, R&D Systems, Minneapolis, USA), TNFA (#43904, BioLegend, San Diego, USA) and KLK7 (E03K0074, Bluegene, Shanghai, China) according to the manufacturer's protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Isolation, Fluorescence, FACS, Reverse Transcription Polymerase Chain Reaction, Marker, Expressing, Recombinant, Control, Two Tailed Test

Journal: Metabolism: clinical and experimental

Article Title: The serine protease KLK7 promotes immune cell infiltration in visceral adipose tissue in obesity.

doi: 10.1016/j.metabol.2025.156239

Figure Lengend Snippet: Fig. 5. Klk7 ablation in macrophages affects migratory capacity and alters immune cell metabolism in obesity. a) Experimental set-up using trans-well inserts to assess the effect of conditioned media (CM) during 6 h, obtained from eWAT explants of HFD-fed KLK7MKO mice and WT littermates, on BMDM migration from chow and HFD-fed KLK7MKO mice and WT littermates. b) ELISA measurement reveals lower levels of MCP1 in conditioned media from eWAT explants of KLK7MKO mice when compared to WT littermates (CM-WT or CM-KO; n = 3 technical replicates from pooled samples). c) Quantification of migration as in (a) reveals a lower migration capacity of BMDM from chow-fed KLK7MKO mice when compared to WT littermates (n = 5 per genotype and diet); and a lower chemo-attractant effect of HFD-fed KLK7MKO derived-CM when compared to HFD-fed WT derived-CM. d) Quantification of BMDM migration as in (a), but with BMDM from HFD-fed mice and using WT-CM reveals a lower migration capacity of BMDM from KLK7MKO mice when compared to WT littermates (n = 4–5 per genotype). e) Cell impedance to continuously measure cell adhesion over 24 h (using a constant current of 64,000 Hz) in BMDM from HFD-fed KLK7MKO mice and WT littermates (n = 5 per genotype). f-h) Time-resolved analysis of the Oxygen Consumption Rate (OCR) (f), quantification of corresponding cellular bioenergetics (basal and maximal respiration, spare respiratory capacity, ATP production, non-mitochondrial respiration, and proton leak, g) and analysis of the time-resolved Extracellular Acidifi cation Rate (ECAR) (h) in BMDM from chow-fed and HFD-fed KLK7MKO mice and WT littermates (n = 3–5 chow-fed and n = 8–9 HFD-fed animals per genotype) measured via the Mito Stress test using a Seahorse extracellular flux analyzer unravels genotypic differences in BMDM from HFD-fed mice. i) Energetic profile map derived from (f) and (g). j) ROS production in BMDM from HFD-fed KLK7MKO mice and WT littermates (n = 8 per genotype) reveals genotypic differences. Ge notypes are illustrated in blue (WT) and green (KLK7MKO), diets by outlined (chow) or filled (HFD) symbols. Significance (p < 0.05) was determined by unpaired two-tailed t-test (b,d,j) and two-way ANOVA with Tukey's post-test (c) or uncorrected Fischer's LSD (e,g). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: We used ELISA to analyze serum levels of insulin (Mouse Insulin ELISA, Mercodia, Uppsala, Sweden), C-peptide (Mouse C-peptide ELISA, ALPCO, Salem, USA), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, USA), adiponectin (Mouse Adiponectin ELISA, AdipoGen, San Diego, USA), MCP1 (Mouse/rat CCL2/JE/MCP-1 Quantikine ELISA, R&D Systems, Minneapolis, USA), TNFA (#43904, BioLegend, San Diego, USA) and KLK7 (E03K0074, Bluegene, Shanghai, China) according to the manufacturer's protocols.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test

Journal: Metabolism: clinical and experimental

Article Title: The serine protease KLK7 promotes immune cell infiltration in visceral adipose tissue in obesity.

doi: 10.1016/j.metabol.2025.156239

Figure Lengend Snippet: Fig. 6. KLK7 expression in human AT and serum is linked to inflammation and immune cell migration. a) KLK7 expression in human VIS and SC AT on the single cell level. Data was from [55]. b) ELISA measurement reveals higher released levels of KLK7 from PBMCs isolated from participants with obesity (n = 7) and cultured over 10 days when compared to PBMCs isolated from participants without obesity (n = 6) from the Tarragona cohort. c) RNA sequencing analysis of KLK7 expression in VIS and SC AT of participants with (n = 1117) or without obesity (n = 26) from the Leipzig Obesity BioBank (LOBB) cohort reveals AT depot specific KLK7 induction in participants with obesity. d) Spearman correlation analysis of KLK7 and body fat in VIS AT (adjusted for age and sex) reveals a positive correlation between the RNA levels of KLK7 and the body fat of the participants from the LOBB cohort (n = 1112). e) Gene set enrichment analysis (GSEA, using KEGG pathways) of genes co- expressed with KLK7 in VIS and SC AT from the LOBB cohort. The top 10 up and down enriched pathways sorted by adj. p and set size are shown. f) Comparison of gene expression of KLK7, inflammatory cytokines (IL1B, IL6, TNFA, RETN and MCP1) and the adipokine LEP in VIS AT in non-obese and obese patients. g) Cor relations of KLK7 and MCP1 expression in VIS (left) and SC (right) AT from the LOBB cohort (adjusted for age and sex) reveal depot-specific differences. h) Cor relation analysis of KLK7 expression with Spearman's correlation in VIS and SC AT from the LOBB cohort with inflammatory cytokines and adipokines (corrected for multiple testing). i) Correlations shared between KLK7 and inflammatory biomarkers in serum from the Leipzig Endocrinology cohort and AT depots from the LOBB cohort reveal markers strongly associated with KLK7. Significance (p < 0.05) was determined by unpaired two-tailed t-test (b) or one-way ANOVA (c).

Article Snippet: We used ELISA to analyze serum levels of insulin (Mouse Insulin ELISA, Mercodia, Uppsala, Sweden), C-peptide (Mouse C-peptide ELISA, ALPCO, Salem, USA), leptin (Mouse Leptin ELISA, Crystal Chem, Downers Grove, USA), adiponectin (Mouse Adiponectin ELISA, AdipoGen, San Diego, USA), MCP1 (Mouse/rat CCL2/JE/MCP-1 Quantikine ELISA, R&D Systems, Minneapolis, USA), TNFA (#43904, BioLegend, San Diego, USA) and KLK7 (E03K0074, Bluegene, Shanghai, China) according to the manufacturer's protocols.

Techniques: Expressing, Migration, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, RNA Sequencing, Comparison, Gene Expression, Two Tailed Test