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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of <t>CXCL12</t> (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by <t>enzyme-linked</t> <t>immunosorbent</t> <t>assay,</t> among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.
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IRG1 / itaconate axis activates Nrf2-TIM4 signaling pathway to alleviate inflammation and oxidative stress. A Western blotting from liver tissues showed that OI promoted the expression of Nrf2, TIM4, HO-1, NQO-1 proteins and decreased the expression of Keap1, p-P65, p-IkB-α, and p-P38 while IRG1-/- reversed these changes. B - C Dual immunofluorescence staining for F480 (red) and TIM4 (green) in mice liver sections. D - E Comparison of immunohistochemistry showing the expression and positivity of Ly6G in the liver tissue of mice in each group. F - J The levels of TNF-α, IFN-γ, IL-1β, IL-10 and IL-6 in serum were detected by <t>ELISA</t> kit. K - L The levels of MDA and total SOD in serum were detected. Data represent the mean ± SD. n = 5, *p < 0.05, ** p < 0.01, ***p < 0.001
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IRG1 / itaconate axis activates Nrf2-TIM4 signaling pathway to alleviate inflammation and oxidative stress. A Western blotting from liver tissues showed that OI promoted the expression of Nrf2, TIM4, HO-1, NQO-1 proteins and decreased the expression of Keap1, p-P65, p-IkB-α, and p-P38 while IRG1-/- reversed these changes. B - C Dual immunofluorescence staining for F480 (red) and TIM4 (green) in mice liver sections. D - E Comparison of immunohistochemistry showing the expression and positivity of Ly6G in the liver tissue of mice in each group. F - J The levels of TNF-α, IFN-γ, IL-1β, IL-10 and IL-6 in serum were detected by <t>ELISA</t> kit. K - L The levels of MDA and total SOD in serum were detected. Data represent the mean ± SD. n = 5, *p < 0.05, ** p < 0.01, ***p < 0.001
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IL-8 secreted by activated neutrophils is suppressed by PFD. A IL-8 secretion by neutrophils measured by <t>ELISA.</t> IL-8 secretion was significantly increased in neutrophils stimulated with CAF-CM compared to non-stimulated neutrophils. PFD administration during neutrophil activation with CAF-CM significantly suppressed IL-8 secretion. B Effect of secretions from activated neutrophils on cancer cell proliferation. The presence of neutrophils significantly increased MIA PaCa-2 proliferation. Mean ± SD is shown. Each dot represents the number of cells per well. Student’s t-test was used. ***, P < 0.001. C Pancreatic cancer cells induce fibroblasts to become iCAFs, and iCAF-CM activates neutrophils, increasing their IL-8 secretion and enhancing the malignant traits of pancreatic cancer cells. Conversely, PFD acts on activated neutrophils, suppressing the malignant traits of pancreatic cancer cells. This figure was created with BioRender.com
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CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of CXCL12 (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of CXCL12 (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

CXCR4 knockdown reduces the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and CXCR4-specific siRNA-transfected (iNSCs treated with CXCR4-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 14 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 15 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and CXCR4-specific siRNA-transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: CXCR4 knockdown reduces the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and CXCR4-specific siRNA-transfected (iNSCs treated with CXCR4-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 14 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 15 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and CXCR4-specific siRNA-transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Knockdown, Activation Assay, Western Blot, Control, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Standard Deviation

Akt knockdown diminishes the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and Akt-specific siRNA–transfected (iNSCs treated with Akt-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 16 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt expression in iNSCs among the four groups (full-length blots are presented in Additional Figure 17 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) expression in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) expression in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific siRNA–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: Akt knockdown diminishes the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and Akt-specific siRNA–transfected (iNSCs treated with Akt-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 16 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt expression in iNSCs among the four groups (full-length blots are presented in Additional Figure 17 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) expression in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) expression in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific siRNA–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Knockdown, Activation Assay, Western Blot, Control, Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Standard Deviation

Akt activation enhances the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 expression in microglia among the control, untransfected (iNSCs note treated with LACs) coculture, control LAC-transfected (iNSCs treated with control LACs) coculture, and Akt-specific LAC-transfected (iNSCs treated with Akt-specific LACs) coculture groups (full-length blots are presented in Additional Figure 18 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) expression in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 19 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (M and O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific LAC–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N and P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; LACs: lentiviral activation particles; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: Akt activation enhances the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 expression in microglia among the control, untransfected (iNSCs note treated with LACs) coculture, control LAC-transfected (iNSCs treated with control LACs) coculture, and Akt-specific LAC-transfected (iNSCs treated with Akt-specific LACs) coculture groups (full-length blots are presented in Additional Figure 18 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) expression in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 19 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (M and O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific LAC–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N and P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; LACs: lentiviral activation particles; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Activation Assay, Western Blot, Expressing, Control, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Standard Deviation

IRG1 / itaconate axis activates Nrf2-TIM4 signaling pathway to alleviate inflammation and oxidative stress. A Western blotting from liver tissues showed that OI promoted the expression of Nrf2, TIM4, HO-1, NQO-1 proteins and decreased the expression of Keap1, p-P65, p-IkB-α, and p-P38 while IRG1-/- reversed these changes. B - C Dual immunofluorescence staining for F480 (red) and TIM4 (green) in mice liver sections. D - E Comparison of immunohistochemistry showing the expression and positivity of Ly6G in the liver tissue of mice in each group. F - J The levels of TNF-α, IFN-γ, IL-1β, IL-10 and IL-6 in serum were detected by ELISA kit. K - L The levels of MDA and total SOD in serum were detected. Data represent the mean ± SD. n = 5, *p < 0.05, ** p < 0.01, ***p < 0.001

Journal: Cell Communication and Signaling : CCS

Article Title: IRG1 /itaconate enhances efferocytosis by activating Nrf2-TIM4 signaling pathway to alleviate con A induced autoimmune liver injury

doi: 10.1186/s12964-025-02075-5

Figure Lengend Snippet: IRG1 / itaconate axis activates Nrf2-TIM4 signaling pathway to alleviate inflammation and oxidative stress. A Western blotting from liver tissues showed that OI promoted the expression of Nrf2, TIM4, HO-1, NQO-1 proteins and decreased the expression of Keap1, p-P65, p-IkB-α, and p-P38 while IRG1-/- reversed these changes. B - C Dual immunofluorescence staining for F480 (red) and TIM4 (green) in mice liver sections. D - E Comparison of immunohistochemistry showing the expression and positivity of Ly6G in the liver tissue of mice in each group. F - J The levels of TNF-α, IFN-γ, IL-1β, IL-10 and IL-6 in serum were detected by ELISA kit. K - L The levels of MDA and total SOD in serum were detected. Data represent the mean ± SD. n = 5, *p < 0.05, ** p < 0.01, ***p < 0.001

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, INF-γ, IL-1β, IL-6, and IL-10 were acquired from DAKEWE Bioengineering ((Beijing, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

IL-8 secreted by activated neutrophils is suppressed by PFD. A IL-8 secretion by neutrophils measured by ELISA. IL-8 secretion was significantly increased in neutrophils stimulated with CAF-CM compared to non-stimulated neutrophils. PFD administration during neutrophil activation with CAF-CM significantly suppressed IL-8 secretion. B Effect of secretions from activated neutrophils on cancer cell proliferation. The presence of neutrophils significantly increased MIA PaCa-2 proliferation. Mean ± SD is shown. Each dot represents the number of cells per well. Student’s t-test was used. ***, P < 0.001. C Pancreatic cancer cells induce fibroblasts to become iCAFs, and iCAF-CM activates neutrophils, increasing their IL-8 secretion and enhancing the malignant traits of pancreatic cancer cells. Conversely, PFD acts on activated neutrophils, suppressing the malignant traits of pancreatic cancer cells. This figure was created with BioRender.com

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer-associated fibroblasts promote pro-tumor functions of neutrophils in pancreatic cancer via IL-8: potential suppression by pirfenidone

doi: 10.1007/s00262-025-03946-z

Figure Lengend Snippet: IL-8 secreted by activated neutrophils is suppressed by PFD. A IL-8 secretion by neutrophils measured by ELISA. IL-8 secretion was significantly increased in neutrophils stimulated with CAF-CM compared to non-stimulated neutrophils. PFD administration during neutrophil activation with CAF-CM significantly suppressed IL-8 secretion. B Effect of secretions from activated neutrophils on cancer cell proliferation. The presence of neutrophils significantly increased MIA PaCa-2 proliferation. Mean ± SD is shown. Each dot represents the number of cells per well. Student’s t-test was used. ***, P < 0.001. C Pancreatic cancer cells induce fibroblasts to become iCAFs, and iCAF-CM activates neutrophils, increasing their IL-8 secretion and enhancing the malignant traits of pancreatic cancer cells. Conversely, PFD acts on activated neutrophils, suppressing the malignant traits of pancreatic cancer cells. This figure was created with BioRender.com

Article Snippet: To evaluate IL-8 levels in neutrophil secretions, culture supernatants were assayed using a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, KE00006) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay