cytokine cocktail m5  (PeproTech)

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: The effect of PEPITEM and SVT[NH-Ethyl] is mirrored in the lymphoid organ repertoire. A) Spleen weight/mouse body weight ratio (expressed as g) evaluated at the experimental endpoint (day 7). Flow cytometry analysis of splenic B) CD4 + , C) CD8 + , D) Th1 (CD4 + IFN-γ + ) and E) Th17 (CD4 + IL-17 + ) subsets, pre-gated on living cells (see flow cytometry strategy reported in Supplementary Fig. 2B and C). Histograms indicate the total positive populations (expressed as x10 6 ) in the different experimental conditions. F) Quantification of T-related cytokines performed by a multi-LEGENDplex ™ analyte flow array (gating strategy is reported in Supplementary Fig. 3) on lymphocytes isolated from skin-draining lymph nodes, following ex vivo stimulation with PMA and ionomycin, presented as a heatmap (colormap:double gradient) and expressed as pg mL −1 . G), H) and I) TNF-α, IL-6 and IL-17A cytokines extrapolated from the heatmap and represented graphically as histograms. Data are presented as mean ± S.D. of n=6 animals in each group. Statistical analysis was conducted by one- or two-way ANOVA followed by Bonferroni’s or Dunnett’s posthoc-test. ## P ≤ 0.01, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs Imiquimod group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: Flow Cytometry, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: PEPITEM Tripeptides and Peptidomimetics: Next-Generation Modulators of Inflammation in Immune-Mediated Conditions

doi: 10.1101/2024.10.14.618151

Figure Lengend Snippet: A) and B) In vitro mechanistic studies on PEPITEM, tripeptides and peptidomimetics. In vitro cytotoxic examination, evaluated by MTT assay, for PEPITEM, tripeptides and peptidomimetics performed on J774A.1 murine macrophage cell lines, following 4 and 24 h of treatment with the selected concentrations (1-30 ng mL −1 ). Dotted lines indicate 75% of cell viability. Data are expressed as cell viability (% of control) and presented as mean ± S.D. of 3 independent experiments. C) and D) IL-6 and TNF-α ELISA assays performed on supernatant of J774A.1 pre-treated with indicated compounds at the concentration of 10 ng mL −1 (highest non-cytotoxic concentration) and then stimulated with LPS (10 µg mL −1 ) for 24 h. E) and F) IL-6 and TNF-α ELISA assays performed on supernatants of NIH-3T3 mouse fibroblasts pre-treated with indicated compounds at the concentration of 10 ng mL −1 and then stimulated with LPS (10 µg mL −1 ) for 24 h. ( C-F ) Data are expressed as pg mL −1 and presented as means ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. # P ≤ 0.05, ### P ≤ 0.001, #### P ≤ 0.0001 vs CTRL group; *P ≤ 0.05, **P ≤ 0.01 vs LPS group. G) Effect of tested compounds on hyperproliferation of M5 cytokines (TNF-α, Oncostatin M, IL-1α, IL-17 and IL-22)-stimulated HaCaT cells, as in vitro psoriasis-like model. HaCaT hyperproliferation (expressed as cells proliferation, % of control) was measured using MTT assay and presented as mean ± S.D. of 3 independent experiments. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s posthoc-test. *P ≤ 0.05 vs M5 group.

Article Snippet: Psoriasis-like keratinocytes model was established by stimulating HaCaT cells with M5 cytokines cocktail consisting of 2.5 ng mL −1 of each TNF-α (210-TA/CF), Oncostatin M (295-OM/CF), IL-1α (200_LA/CF), IL-17 (317-ILB) and IL-22 (782-IL/CF, all from R&D System, Italy) as previously described , .

Techniques: In Vitro, MTT Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cellular & molecular immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis.

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Fig. 5 TWEAK promotes neutrophil expression of LCN2 but enhances inflammation in keratinocytes by inducing MMP9 expression. A Relative mRNA expression level of Lcn2, Tnfrsf12a, Tgfb1, Il6, Tnfa, Il1b, Krt5, Krt10, Krt17, 24p3r, Mc4r in mouse skin treated with TWEAK was detected by RT-qPCR. B Immunostaining of LCN2 and Ly6G were performed on paraffin sections of IMQ mice model with or without dorsally topical application of rmTWEAK (20 μg/mL, prepared in PBS). Unpaired two-tailed t test. C Immunofluorescence assays were used to detect LCN2 expression in neutrophils after TWEAK stimulation. Bar = 20 μm. D RT-qPCR was conducted to detect Lcn2 mRNA level in neutrophils treated with TWEAK. E LCN2 levels in neutrophil supernatants after TWEAK stimulation for 24 h were measured by ELISA. F Proteomic analysis was used to further investigate the protein changes and possible pathways following TWEAK action on keratinocytes. G After treating with TWEAK on primary keratinocyte with or without M5 cytokines for 48 h, LCN2 and 24P3R protein levels in keratinocytes were detected by Western blotting. H After the TWEAK stimulus, proteins such as MMP9 and TRAF2 were detected by Western blotting. Data from immunofluorescence assays and ELISA assay are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells. ANOVA was used for comparison between groups: *P < 0.05, **P < 0.01, and ***P < 0.001. ns not significant

Article Snippet: Subsequently, rmLCN2 (0–500 ng/mL; R&D Systems, Minneapolis, MN) and rmTWEAK (100 ng/mL; R&D Systems, Minneapolis, MN) were administered either separately or simultaneously, followed by stimulation with the M5 cocktail (including TNF-α, oncostatin M, IL-17A, IL-1α, IL-22 each at 10 ng/ mL; ProteinTech, Wuhan, China) for 48 hours [17].

Techniques: Expressing, Quantitative RT-PCR, Immunostaining, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison