quantikine elisa kit  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: The angiogenic potential of the co-culture. The human HT1080 fibroblast cell line (3 × 10 5 cells) or the human U937 monocytic-like cell line (3 × 10 5 cells) were plated in 24-well plates alone or in co-culture (U937 cells were plated in inserts with 0.4-μm pores size) and incubated for 48 h in the presence of TNFα (1 ng/ml). Then, supernatants were collected, and the concentrations of (A) CD147, (B) VEGF, (C) MMP-9, (D) endostatin, (E) Tsp-1, and (F) TIMP-1 were assessed by ELISA (n = 8). Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test. (G, H) EaHy926 cells (2 × 10 4 cells) were grown to confluency, and then a scratch was applied using the edge of a tip. Detached cells were washed away, and supernatants derived from the experimental groups were diluted 1:2 with serum-starvation medium and applied onto the endothelial layer, with or without the presence of the rabbit anti-human CD147 antibody (h161-pAb, 2 ng/ml). Images were taken at the beginning of the experiment (0 h) and after 20 h. (G) Representative images and (H) the measured distance between the two sides of the scratch at 20 h subtracted from the distance at 0 h (n = 8–9 per group) are shown. Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: To determine the concentrations of the pro- and anti-angiogenic factors, human DuoSet ELISA kits for CD147, MMP-9, MMP-2, VEGF, endostatin, thrombospondin-1, TIMP-1, and TIMP-2 were used according to the manufacturer’s instructions (R&D Systems).

Techniques: Co-Culture Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: CD147 regulates the accumulation of endostatin. HT1080 cells (3 × 10 4 cells) were incubated alone or in co-culture with U937 cells (3 × 10 4 cells) for 48 h in the presence of TNFα (1 ng/ml). (A) The cells were incubated alone, with the anti-CD147 antibody (h161-pAb, 2 ng/ml) or with an irrelevant antibody (rabbit IgG, 2 ng/ml). After 48-h incubation, supernatants were collected, and the concentrations of endostatin (n = 6) were determined by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test. (B) The human HT1080 fibroblast cell line (10 5 cells) was transfected with two CD147 siRNA molecules (10 nM each) and left for 24 h in full medium. Then, the medium was replaced with serum-starvation medium (antibiotics-free) with TNFα (1 ng/ml), and U937 cells (10 5 cells) were added in inserts (0.4-μm pore size). After 48-h incubation in co-cultures, supernatants were collected, and the concentrations of endostatin (n = 8) were assessed using ELISA. Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Bonferroni’s post-hoc test. (C) HT1080 cells (3 × 10 4 cells), U937 cells (3 × 10 4 cells), and their co-culture were incubated in serum-starvation medium with TNFα (1 ng/ml), in the presence of the human recombinant CD147 (300 ng/ml) or the Fc fragment (300 ng/ml) for 48 h. After incubation, supernatants were collected, and the concentrations of endostatin (n = 6) were assessed by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: To determine the concentrations of the pro- and anti-angiogenic factors, human DuoSet ELISA kits for CD147, MMP-9, MMP-2, VEGF, endostatin, thrombospondin-1, TIMP-1, and TIMP-2 were used according to the manufacturer’s instructions (R&D Systems).

Techniques: Incubation, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Transfection, Pore Size, Recombinant

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: CD147 hormetically regulates endostatin. The HT1080 fibroblast cell line (3 × 10 5 cells) was cultured alone or co-cultured with U937 cells (3 × 10 5 cells) in serum-starvation medium in the presence of TNFα (1 ng/ml). (A, B) Supernatants were collected at different time points, and the concentrations of (A) endostatin (n = 6) and (B) VEGF (n = 6) were assessed by ELISA. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test. (C–E) Increasing concentrations of recombinant CD147 were added, and the cultures were incubated for 48 h. Supernatants were collected, and concentrations of (C) endostatin, (D) TIMP-1 (n = 6), and (E) MMP-9 (n = 6) were assessed using ELISA. **, p < 0.01; ***, p < 0.001 relative to unstimulated HT1080 cells; $, p < 0.05, $$$, p < 0.001 relative to unstimulated co-culture; &, p < 0.05, &&, p < 0.01, &&&, p < 0.001 relative to HT1080 cells in each concentration. Data are presented as means ± SE and were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test.

Article Snippet: To determine the concentrations of the pro- and anti-angiogenic factors, human DuoSet ELISA kits for CD147, MMP-9, MMP-2, VEGF, endostatin, thrombospondin-1, TIMP-1, and TIMP-2 were used according to the manufacturer’s instructions (R&D Systems).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Co-Culture Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: Proteasome 20S and MMP-9 are involved in endostatin generation. The HT1080 cells (3 × 10 4 cells) in single cultures or in co-culture with U937 cells (3 × 10 4 cells) were incubated in serum-starvation medium in the presence of TNFα (1 ng/ml), together with different protease inhibitors that were added at the following concentrations: leupeptin 1 μM, pepstatin A 5 μM, MG132 1 μM, AM114 1 μM, phenantroline 40 μM, NSC405020 100 μM, MMP-9 inhibitor I 5 nM, and disulfiram 20 μM. Endostatin levels were evaluated by ELISA in supernatants derived from (A) HT1080 single cultures and (B) co-cultures (n = 12). Data are presented as means ± SE and were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: To determine the concentrations of the pro- and anti-angiogenic factors, human DuoSet ELISA kits for CD147, MMP-9, MMP-2, VEGF, endostatin, thrombospondin-1, TIMP-1, and TIMP-2 were used according to the manufacturer’s instructions (R&D Systems).

Techniques: Co-Culture Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Soluble CD147 regulates endostatin via its effects on the activities of MMP-9 and secreted proteasome 20S

doi: 10.3389/fimmu.2024.1319939

Figure Lengend Snippet: The effect of disease on the serum levels of RA patients. Serum samples from patients with active RA were collected (RA, n = 23) and the concentrations of (A–C, E–G) the pro- and anti-angiogenic factors were compared to those in the serum of healthy volunteers (HC, n = 25). (D, H) Additionally, the activities of proteasome 20S and MMP-9 were evaluated in the serum samples. Data are presented as median values (red) and were analyzed using the non-parametric Mann–Whitney test. (I, J) The functional wound assay was carried out using cells of the confluent human endothelial cell line EaHy926 that were scratched. Serum samples from RA patients (n = 12) or controls (n = 12) were diluted 1:2 in full medium and applied onto the endothelial layer, with or without the presence of the rabbit anti-human CD147 antibody (h161-pAb, 2 ng/ml). Images were taken at the beginning of the experiment (0 h) and after 20 h. The distance between the two sides of the scratch at 20 h was subtracted from the distance at 0 h, to calculate the net distance that endothelial cells migrated to. Data are presented as means ± SE and were analyzed using the one-way ANOVA test followed by Bonferroni’s post-hoc test. (K–P) The human HT1080 fibroblast cell line (3 × 10 4 cells) was seeded in 96-well plates and incubated in serum-starvation medium for 48 h in the presence of TNFα (1 ng/ml) and the serum samples (diluted 1:2) obtained from controls or RA patients and with or without h161-pAb (2 ng/ml). Then, supernatants were collected, and the concentrations of (K) endostatin (n = 13), (L) MMP-9 (n = 13), (N) Tsp-1 (n = 13), and (O) TIMP-1 (n = 13) were determined by ELISA. The activities of (M) MMP-9 (n = 13) and (P) proteasome 20S (n = 13) were measured. Data are presented as means ± SE and were analyzed using the one-way ANOVA test followed by Bonferroni’s post-hoc test.

Article Snippet: To determine the concentrations of the pro- and anti-angiogenic factors, human DuoSet ELISA kits for CD147, MMP-9, MMP-2, VEGF, endostatin, thrombospondin-1, TIMP-1, and TIMP-2 were used according to the manufacturer’s instructions (R&D Systems).

Techniques: MANN-WHITNEY, Functional Assay, Incubation, Enzyme-linked Immunosorbent Assay