anti c3ar  (Hycult Biotech)

Journal: BMC Immunology

Article Title: Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor

doi: 10.1186/1471-2172-9-29

Figure Lengend Snippet: Anaphylatoxin receptors are expressed on activated MC . Precursor cell-derived murine MC cultured with IL-3 and SCF were treated or not (Ø) with ionomycin, PMA, or Ag (DNP-albumin, following a 24 h preincubation period with IgE anti-DNP) for 24 h (A) or for 1 h and 4 h, respectively (B). Subsequently, MC were stained by indirect immunofluorescence and analyzed by FACS. Filled histograms indicate staining with rat IgG1 control mAb, open histograms (solid lines) with mAb 1240 against murine C5aR, and open histograms (dotted lines) with mAb 1G4 against murine C3aR. One representative experiment each of at least 3 is shown.

Article Snippet: The following primary mAb were used at a concentration of 5 μg/ml: anti-murine C5aR mAb 1240 (rat IgG1), anti-murine C3aR mAb 1G4 (rat IgG1), isotype control mAb rat IgG1 (BD Biosciences).

Techniques: Derivative Assay, Cell Culture, Staining, Immunofluorescence

Journal: BMC Immunology

Article Title: Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor

doi: 10.1186/1471-2172-9-29

Figure Lengend Snippet: Activated MC respond to C5a in vivo . Precursor cell-derived murine MC cultured with IL-3 and SCF were treated or not (Ø) with ionomycin, PMA or Ag (DNP-albumin, following a 24 h preincubation period with IgE anti-DNP) for different periods as indicated. Subsequently, MC were labeled with PKH26. In (C, D), labeled MC were additionally preincubated at 37°C without (Ø) or with C5a (2 μg) to induce receptor desensitization. In (E), labeled MC were preincubated on ice without (Ø) or with mAb 1240 (20 μg, rat IgG1) to block C5aR, or with mAb 1G4 (20 μg, rat IgG1), as a control. Thereafter, labeled MC were injected i.v. into syngeneic BALB/c mice together with C5a (10 μg) i.p.. After 4 h (A, B, E) or 24 h (C, D, F), peritoneal cells were harvested and labeled migratory cells identified by FACS analysis. Mean values (± SEM) of 3 (A, B, D) or 4 (C, E, F) independent experiments each are shown.

Article Snippet: The following primary mAb were used at a concentration of 5 μg/ml: anti-murine C5aR mAb 1240 (rat IgG1), anti-murine C3aR mAb 1G4 (rat IgG1), isotype control mAb rat IgG1 (BD Biosciences).

Techniques: In Vivo, Derivative Assay, Cell Culture, Labeling, Blocking Assay, Injection

Journal: BMC Immunology

Article Title: Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor

doi: 10.1186/1471-2172-9-29

Figure Lengend Snippet: C5aR upregulation is independent of SCF treatment . Precursor cell-derived murine MC cultured with IL-3 were treated or not (Ø) with ionomycin, PMA, or Ag (DNP-albumin, following a 24 h preincubation period with IgE anti-DNP) for 24 h (A) or 4 h (B). In (A), MC were stained by indirect immunofluorescence and analyzed by FACS. Filled histograms indicate staining with rat IgG1 control mAb, open histograms (solid lines) with mAb 1240 against murine C5aR, and open histograms (dotted lines) with mAb 1G4 against murine C3aR. In (B), MC were labeled with PKH26 and injected i.v. into syngeneic BALB/c mice together with C5a (10 μg) i.p.. 24 h later, peritoneal cells were harvested and labeled migratory cells identified by FACS analysis. One representative experiment of 3 (A) and mean values (± SEM) of 4 independent experiments each (B) are shown.

Article Snippet: The following primary mAb were used at a concentration of 5 μg/ml: anti-murine C5aR mAb 1240 (rat IgG1), anti-murine C3aR mAb 1G4 (rat IgG1), isotype control mAb rat IgG1 (BD Biosciences).

Techniques: Derivative Assay, Cell Culture, Staining, Immunofluorescence, Labeling, Injection

Journal: BMC Immunology

Article Title: Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor

doi: 10.1186/1471-2172-9-29

Figure Lengend Snippet: Activation of peritoneal MC induces C5aR upregulation . Peritoneal MC from BALB/c mice were purified by a negative selection technique and cultured with ionomycin (A) or Ag (DNP-albumin, following a 24 h preincubation period with IgE anti-DNP) (B) for different periods as indicated. Subsequently, MC were stained by indirect immunofluorescence and analyzed by FACS. Gates were set on CD117 + MC (A, B). In (C), untreated peritoneal lavage cells were stained by indirect immunofluorescence and gates set on F4/80 + macrophages. Filled histograms indicate staining with rat IgG1 control mAb, open histograms (solid lines) with mAb 1240 against murine C5aR, and open histograms (dotted lines) with mAb 1G4 against murine C3aR. One representative experiment each of 3 is shown.

Article Snippet: The following primary mAb were used at a concentration of 5 μg/ml: anti-murine C5aR mAb 1240 (rat IgG1), anti-murine C3aR mAb 1G4 (rat IgG1), isotype control mAb rat IgG1 (BD Biosciences).

Techniques: Activation Assay, Purification, Selection, Cell Culture, Staining, Immunofluorescence

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: Activation of complement C3-C3aR/ITGAM pathway in BCCAO rats. A Quantitative RT-PCR analysis of the expression of C1qa, C1qb, C4b, C3 , C3ar , and Itgam in the striatum of control and BCCAO rats at day 7, 14, and 28 after BCCAO surgery. The values are normalized to those of the control group. n = 3-7 in each group. B Western blots and quantification for C3, C3aR, ITGAM, and β-actin in the striatum of control and BCCAO rats at day 7, 14, and 28 after surgery. C Representative images and quantification of complement C3 puncta (red) deposition on myelin (MBP + , green) in the striatum of BCCAO and control rats. n = 3-4 animals in each group at day 7, 14, and 28 after surgery. Scale bar=10 μm. The data are shown as the mean ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant; the BCCAO group vs. the control group.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: Genetic deletion of C3ar1 attenuates microglia activation and reverses white matter injury in BCAS mice. A Representative images and quantification of C3aR (green) and Iba-1 (red) double-positive cells and microglia cells (Iba-1 + cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=25 μm. B Western blots and quantification for CD86, iNOS, MBP and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. C Representative images and quantification of damaged axon (SMI32 + , red) relative to myelin (MBP + , green) and mature oligodendrocyte (APC + cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=50 μm. D Western blots of total- and phospho-STAT3 (pSTAT3) and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. E and F Quantification of total STAT3/ β-actin ( E ) and phospho-STAT3/STAT3 ( F ) levels in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. The data are shown as the mean ± SD. n = 3-4 animals in each genotype group. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Activation Assay, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: C3aR inhibition suppresses microglial activation and microglia redistribution to myelin in BCCAO rats. A Western blots and quantification for CD86, iNOS, and β-actin in the striatum of the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 3-6 in each group. B Representative images of microglia cells (Iba-1 + cells, red) contacting with myelin (MBP + , green) in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. Scare bar=50 μm. n= 3-4 in each group. C-D Quantification of the proportion of microglia cells adhered to myelin relative to the number of myelin fibers ( C ) and the number of microglia cells ( D ). The data are shown as the mean ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant; the BCCAO group vs. control group. C3aRA: C3aR antagonist.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Inhibition, Activation Assay, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: C3aR inhibition prevents behavioral deficits and white matter injury in BCCAO rats. A Five-day spatial learning performance measured as the latency to reach the platform in the Morris water maze test in sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n= 12-15 animals for each group. B Spatial memory performance measured as the number of entries into the platform quadrant and the percentage of time spent in the platform quadrant in the Morris water maze test in the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups, n = 12-15 animals in each group. C Spatial memory performance measured as discrimination time in the new object recognition test in the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 4-6 animals for each group. D Western blots and quantification for myelin basic protein (MBP) and β-actin in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. E - F Representative images and quantification of damaged axon (SMI32 + , red) relative to myelin (MBP + , green) ( E ) and mature oligodendrocyte (APC + cells, red) ( F ) in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 3-4 animals in each group. Scale bar=50 μm. The data are shown as the mean ± SD. **, p < 0.01; *, p < 0.05; the BCCAO group vs. control group. C3aRA: C3aR antagonist.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Inhibition, Western Blot