elisa kit  (R&D Systems)

Journal: Inflammopharmacology

Article Title: Crosstalk between ferroptosis and NLRP3, a possible therapeutic target in experimentally-induced rheumatoid arthritis: role of P2Y12R inhibition in modulating P53/SLC7A11/ALOX15 signaling

doi: 10.1007/s10787-025-01841-8

Figure Lengend Snippet: Effect of ticagrelor on pro-inflammatory chemokines and NLRP3 in AIA rat model. A RANTES/CCL5. B MIP-1α/CCL3, C Eotaxin-3 /CCL26 D NLRP3 using ELISA analysis. First Bar represents the control group, Second Bar represents the AIA group, Third Bar represents the Ticagrelor group and Fourth Bar represents the Ticagrelor + AIA group. E Correlation analysis between NLRP3 and all chemokine markers. Ticagrelor was given orally (30 mg/kg) for 21 days, AIA was induced by FCA (0.1 ml single dose S.C.). The left hind paws of rats from each group were separated on (day 22) then kept at -80°C for RANTES/CCL5, MIP-1α/CCL3, Eotaxin-3/CCL26 and NLRP3 ELISA analysis. Two-way ANOVA with Tukey-Kramer post hoc testing was employed for statistical analysis. Data are provided as means ± SD; p ≤ 0.05. (ns) non-significance, *p < 0.05. ****p < 0.0001. Correlation analysis was implemented based on Pearson’s correlation coefficient

Article Snippet: Minces of ankle tissues were homogenized in ice- phosphate-buffered saline (ice-PBS) (usually 10 mg tissue in 100 μl of PBS), and then tissue homogenates were subjected according to the ELISA kit with the source and catalog number to estimate the following parameters: SLC7A11 (Biorbyt, Cat. No: orb781117, Cambridge, UK), GPX4 (MyBioSource, Cat. No: MBS069787, US), FTH1 (LSBio, Cat. No: LS- F33593 , US), ACSL4 (MyBioSource, Cat. No: MBS3809786, US), ALOX15 (MyBioSource, Cat. No: MBS103451, US), and inflammatory chemokines like RANTES/CCL5 (R&D Systems QuantikineTM ELISA, MMR00, US), macrophage inflammatory protein-1α (MIP-1α)/CCL3 (Abcam, ab213916, Cambridge, UK), eotaxin-3/CCL26 (MyBioSource, Cat. No: MBS263571, US), and NLRP3 inflammasome (MyBioSource, Cat. No: MBS7255410, US).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 1. Changes in CCL5 and HMGB1 protein levels changes at lesion sites following rat SCI. (A) ELISA measurement of CCL5 protein levels at lesion sites following SCI at 0 d, 1 d, 4 d and 7 d, respectively. (B) Western blot analysis of HMGB1 expression following SCI at 0 d, 1 d, 4 d and 7 d. (C) Quantification data are shown in (B). Quantities were normalized to endogenous β-actin. (D) ELISA analysis of CCL5 protein levels at lesion sites at 0 d, 1 d, 4 d and 7 d following with or without intrathecal injection of 10 µl of an HMGB1 neutralizing antibody (HMGB1 Ab, 50 µg/kg). (E) Immunofluorescence staining revealed the colocalization of CCL5 with GFAP-positive astrocytes before or after SCI at 4 d with or without the intrathecal injection of 10 µl of HMGB1 Ab (50 µg/kg). The rectangle indicates the region magnified. Arrowheads indicate positive signals. n = 6. Scale bars, 50 μm in (E). The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Immunofluorescence, Staining, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 2. Examination of CCL5 production in astrocytes following stimulation with rHMGB1. (A, B) Purified primary astrocytes were stained with GFAP and Hoechst 33,342, and the purity was greater than 90%. Scale bar, 50 μm. (C, D) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following stimulation with 0–2.5 µg/mL rat recombinant HMGB1 (rHMGB1) for 24 h, respectively. (E, F) ELISA assay was used to determine the production of CCL5 in the lysates and supernatants following primary astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of 2.5 µg/ml HMGB1 Ab, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 3. Effects of RAGE, TLR-2, or TLR-4 interference on HMGB1-induced CCL5 production in astrocytes. (A, B) Cell lysates and supernatants were tested by ELISA for the production of CCL5, following astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of the RAGE inhibitor FPS-ZM1 (2 µM) for 24 h. (D, E) ELISA was used to determine CCL5 protein levels in lysates and supernatants from astrocytes after stimulation with 0.5 µg/ml rHMGB1 in the presence of the TLR2 inhibitor C29 (10 µM) for 24 h. (G, H) CCL5 levels in lysates and supernatants of astrocytes were determined by ELISA after the astrocytes were treated with the TLR4 inhibitor TAK-242 (2 µM) in the presence of 0.5 µg/ml rHMGB1 for 24 h. (C,F, I) CCK8 assay of the effects of FPS-ZM1, C29, or TAK-242 on the viability of astrocytes. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 4. Determination of CCL5 synthesis-related protein levels in astrocytes following stimulation with rHMGB1. (A) Western blot analysis of the phosphorylation of the ERK, JNK, P38 kinase and p65NF-κB proteins after astrocytes were treated with 0–2.5 µg/mL rHMGB1 for 24 h. (B-E) Quantification data are shown in (A). Quantities were normalized to endogenous β-actin. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Western Blot, Phospho-proteomics, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 5. Effects of the inhibition of MAPK/NF-κB signaling on the HMGB1-induced production of CCL5 from astrocytes. (A, B) ELISA was used to determine the effects of 10 µM ERK inhibitor PD98059, 10 µM JNK inhibitor SP6001251 or 10 µM P38 inhibitor SB203580 in the presence of 0.5 µg/ml rHMGB1 for 24 h on CCL5 protein production levels in lysates and supernatants of astrocytes. (C) Western blot analysis of the activation of p65NFκB protein after astrocyte treatment with 10 µM ERK inhibitor PD98059 or 10 µM JNK inhibitor SP6001251 for 24 h in the presence of 0.5 µg/ml rHMGB1. (D) Quantification data are shown in (C). Quantities were normalized to endogenous β-actin. (E, F) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following challenge with 10 µM SN50 for 24 h in the presence of 0.5 µg/ml rHMGB1, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 6. Effects of HMGB1-mediated astrocytic CCL5 on the migration of BV2 microglia cells or RAW 264.7 macrophage cells in vitro. (A) Illustration of the BV2 or RAW 264.7 cells with rCCL5 coculture model. (B) Transwell assay analysis of migration the ability of BV2 or RAW 264.7 cells co-incubated with 0.1 µg/ml rCCL5 for 48 h. (C) and (D) Quantification data are shown in (B). (E) Illustration of the BV2 or RAW 264.7 cells and ACM coculture model. (F) Migration assay of BV2 or RAW 264.7 cells cocultured with ACM. ACM were prepared from astrocytes exposed to 0.5 µg/ml rHMGB1 for 24 h, followed by the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. (G) and (H) Quantification data are shown in (F). Scale bars, 100 μm in (B) and (F). The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Migration, In Vitro, Transwell Assay, Incubation, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 7. Effects of HMGB1-induced astrocytic CCL5 on microglia/macrophage polarization. (A) and (B) qRT-PCR was used to determine the expression of M1 markers (CD86, iNOS and TNF-a) and M2 markers (CD206, Arg1 and Ym1) after BV2 or RAW 264.7 cells incubated with 0.1 µg/ml rCCL5 for 24 h. (C) and (D) The expression levels of M1 and M2 markers were determined by qRT-PCR following BV2 or RAW 264.7 cells cocultured with ACM for 24 h. ACM was prepared by astrocytes challenge with 0.5 µg/ml rHMGB1 for 24 h, after which the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 8. Immunofluorescence of microglia/macrophage and locomotor function assessment after the inhibition of HMGB1 or CCL5 by neutralizing antibodies in rat SCI. (A) Immunofluorescence of IBA1- or CD68- positive cells at 4 d following SCI after neutralizing antibody application by intrathecal injection of 10 µl of neutralizing antibodies (HMGB1 Ab, 50 µg/kg), CCL5 (CCL5 Ab, 50 µg/kg) or normal rabbit IgG (IgG, 50 µg/ kg). n = 6. Scale bars, 50 μm. (B, C) Quantitative analysis of the intensity of IBA1- or CD68-labeled cells in (A), respectively. (D) HE staining of the injured spinal cord at 21 d after administration of 10 µl of HMGB1, CCL5 neutralizing antibodies or normal rabbit IgG. n = 6. Scale bars, 500 μm. (E) Quantification data are shown in (D). (F) Basso, Beattie, and Bresnahan (BBB) locomotor scale scores for hindlimb motor function in rats at 0 d, 7 d, 14 d, 21 d and 28 d following the intrathecal administration of the HMGB1 Ab, CCL5 Ab or normal rabbit IgG. n = 6. The experiments were performed in triplicate. The error bars represent the standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Immunofluorescence, Inhibition, Injection, Labeling, Staining, Standard Deviation

Journal: Scientific reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury.

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Fig. 9. Mechanistic diagram of HMGB1-mediated astrocytic CCL5 contributes to microglia/macrophages activation and recruitment.

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Activation Assay