rabbit anti rat ccl2 polyclonal igg antibody  (Bio-Rad)

Journal: ASN Neuro

Article Title: Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

doi: 10.1042/an20120048

Figure Lengend Snippet: Figure 3 TNFα increases CCL2 mRNA content in primary and immortalized PNMEC cultures (A) Primary PNMEC cultures were incubated in the absence (media) or pres- ence of TNFα (10 ng/ml) for 3 h. (B, C) Immortalized PNMEC cultures were treated with (B) 0–100 ng/ml TNFα for 2 h or (C) 0–6 h with 10 ng/ml TNFα, as indicated. Relative changes in CCL2 mRNA content were quantified by qRT-PCR (quantitative real-time PCR). Data shown are the GAPDH-normal- ized fold changes from two separate experiments (n = 4–9 cultures) and expressed as means+−S.E.M. #, P < 0.05; unpaired Student’s t-test. *, P < 0.05; ** P < 0.01; one-way ANOVA with Dunnett’s post-hoc analysis.

Article Snippet: Washed membranes were blocked and incubated overnight at 4 ◦C in the presence of primary antibody (1:10 dilution of mouse anti-rat large T antigen (provided by Dr. P. Jat), 1:4000 dilution of rabbit anti-rat CCL2 polyclonal, or 1:100 dilution of mouse anti-rat ICAM-1 monoclonal antibody (Serotec).

Techniques: Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: ASN Neuro

Article Title: Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

doi: 10.1042/an20120048

Figure Lengend Snippet: Figure 4 TNFα increases CCL2 protein content in primary and immortalized PNMEC cultures Intracellular CCL2 content in (A) primary or (B) immortalized PNMEC cultures incubated in the absence (media) or presence of TNFα (10 ng/ml) for 4 h was quantified by cell-based ELISA or immunoblot, respectively. (C, D) Immortalized PNMEC cultures were cultured with (C) 0–100 ng/ml TNFα for 4 h or (D) 0–24 h with 10 ng/ml TNFα, as indicated, and intracellular CCL2 protein was quantified by cell-based ELISA. Data shown are results from 2–3 separate experiments (A, n = 12–14 cultures; C, D, n = 6–10 cultures). Immunoblot data shown in (B) are from a single experiment performed in duplicate. (A) #, P < 0.0001 unpaired Student’s t-test; (C) *, P < 0.05; ** P < 0.01; one-way ANOVA with Dunnett’s post-hoc analysis. (D) *, P < 0.001; two-way ANOVA with Bonferroni’s post-hoc analysis.

Article Snippet: Washed membranes were blocked and incubated overnight at 4 ◦C in the presence of primary antibody (1:10 dilution of mouse anti-rat large T antigen (provided by Dr. P. Jat), 1:4000 dilution of rabbit anti-rat CCL2 polyclonal, or 1:100 dilution of mouse anti-rat ICAM-1 monoclonal antibody (Serotec).

Techniques: Incubation, In-Cell ELISA, Western Blot, Cell Culture

Journal: ASN Neuro

Article Title: Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

doi: 10.1042/an20120048

Figure Lengend Snippet: Figure 7 TNFα promotes secretion of CCL2 from primary and immortalized PNMEC cultures (A) Content of CCL2 secreted from primary PNMEC cultures incubated in the absence (media) or presence of TNFα (10 ng/ml) for 4 h. (B, C) Immortalized PNMEC cultures were treated with (B) 0–100 ng/ml TNFα for 4 h or (C) 0–8 h with 10 ng/ml TNFα, as indicated, and the content of CCL2 released into the cell culture media was quantified by ELISA. Data shown are the means+−S.E.M. (n = 3–6 cultures). (A) #, P < 0.0001, Student’s t-test. (B, C) *; P < 0.05; **; P < 0.01, one-way ANOVA with Dunnett’s post-hoc analysis.

Article Snippet: Washed membranes were blocked and incubated overnight at 4 ◦C in the presence of primary antibody (1:10 dilution of mouse anti-rat large T antigen (provided by Dr. P. Jat), 1:4000 dilution of rabbit anti-rat CCL2 polyclonal, or 1:100 dilution of mouse anti-rat ICAM-1 monoclonal antibody (Serotec).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: ASN Neuro

Article Title: Tumour Necrosis Factor α Enhances CCL2 and ICAM-1 Expression in Peripheral Nerve Microvascular Endoneurial Endothelial Cells

doi: 10.1042/an20120048

Figure Lengend Snippet: Figure 8 Secreted CCL2 promotes migration of THP-1 monocytes across PNMEC culture monolayers Immortalized PNMEC cultures were incubated in the absence (media) or presence of TNFα (10 ng/ml) for 4 h, and conditioned cell- culture medium was used to assay for THP-1 monocyte chemotaxis across PNMEC monolayers. (A) Total migrated cells in response to 2–6 h treatment with medium or TNFα-conditioned medium. (B) Total migrated cells in response to 4 h treatment with medium or TNFα-conditioned medium supplemented without (control) or with an CCL2 neutralizing antibody (α-CCL2) or with heat-denatured anti-CCL2 antibody. Data shown are the means+−S.E.M. (n = 3–7 cultures). (A) *, P < 0.01, two-way ANOVA with Bonferroni’s post-hoc analysis. (B) One-way ANOVA with Bonferroni’s post-hoc analysis.

Article Snippet: Washed membranes were blocked and incubated overnight at 4 ◦C in the presence of primary antibody (1:10 dilution of mouse anti-rat large T antigen (provided by Dr. P. Jat), 1:4000 dilution of rabbit anti-rat CCL2 polyclonal, or 1:100 dilution of mouse anti-rat ICAM-1 monoclonal antibody (Serotec).

Techniques: Migration, Incubation, Cell Culture, Chemotaxis Assay, Control