anti rat c3 c3b  (Hycult Biotech)

Journal: Blood Advances

Article Title: Antibody-mediated immune suppression by antigen modulation is antigen-specific

doi: 10.1182/bloodadvances.2018018408

Figure Lengend Snippet: Antibody and complement deposition is detectable on HOD × KEL RBC posttransfusion, with clearance of HOD × KEL RBC occurring in anti-KEL immunized recipients. (A) HOD × KEL RBC were labeled before transfusion into PBS (no antibody), anti-KEL–, or anti-HEL–treated WT mice. (B) IgG deposition was measured by direct antiglobulin test at 1, 2, and 24 hours posttransfusion. Total C3 (C) and active complement (C3b/iC3b) (D) was measured at 1, 2, and 24 hours posttransfusion. (E) RBC survival of HOD × KEL+ compared with HOD × KEL− RBC at various times posttransfusion. Means ± standard deviation (SD) shown. (A-D) ****P < .0001, ***P < .001, **P < .01, and not significant (ns) by 1-way ANOVA with Tukey multiple comparison test. (E) ****P < .0001 and ns by 2-way ANOVA with Dunnett multiple comparisons test. Data shown include 5 mice per group and are representative of 3 independent experiments. DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DiO, 3,3′-dioctadecyloxacarbocyanine perchlorate; FSC, forward scatter; MFI, mean fluorescence intensity; SSC, side scatter.

Article Snippet: 24 , 38 - 40 Briefly, RBC were stained with APC anti-mouse IgG (Jackson Immunoresearch, West Grove, PA) for assessment of bound antibody, or with biotin anti-mouse complement component 3 (C3; Cedarlane, Burlington, NC) for assessment of total complement or biotin anti-mouse C3b (Cedarlane) for detection of active complement, followed by streptavidin APC (BD, Franklin Lakes, NJ).

Techniques: Labeling, Direct Antiglobulin Test, Standard Deviation, Fluorescence

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A) C3 fragment staining (false color green) and (B) C9 staining (brown) of kidney biopsies of patients with SCD nephropathy, performed by immunofluorescence of frozen tissue and immunohistochemistry of paraffin-embedded tissues, respectively. A normal protocol kidney allograft biopsy performed at 3 months was used as a negative control and a biopsy of a patient with acute humoral rejection was used as a positive control for the staining. (C) C3b/iC3b (false color green) and C5b-9 (false color red) staining of kidney sections of HbAA and HbSS mice. Quantification of the C3 and C5b-9 staining in HbSS (n ≥ 7 mice per group) and HbAA (n ≥ 4 mice per group) mouse kidney glomeruli. (D) C3b/iC3b (false color green) and C5b-9 (false color red) staining of kidney sections of SAD mice and WT littermates. Quantification of the C3 and C5b-9 staining in kidney glomeruli (n ≥ 7 mice per group). (E) Double staining of SAD and HbSS mouse kidney sections for C3b/iC3b (false color green) and endothelial marker CD31 (false color red). The merge image indicates colocalization in orange. One glomerulus was focused on. *P < 0.05; **P < 0.005; ***P < 0.001, Mann-Whitney test. Values are shown as box plots with median and minimum/maximum points. Scale bar: 50 μm. Original magnification, ×26.

Article Snippet: Complement deposition was studied using rat anti-mouse C3 fragments (C3bi/C3b/C3c) IgG1 (Hycult Biotech, HM1065), visualized with goat anti-rat-AF488 (Invitrogen, A-11006) and rabbit anti-mouse C5b-9 (Abcam, ab55811), and visualized with goat anti-rabbit IgG-AF488 (Invitrogen, A-11008).

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Negative Control, Positive Control, Double Staining, Marker, MANN-WHITNEY

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A) C3b/iC3b (false green color) staining of frozen kidney sections of mice injected with PBS or PHZ and sacrificed at 6 hours. Quantification of C3 fragment staining in glomeruli (n ≥ 5 mice per group). (B) Double staining of PHZ-injected mouse kidney sections for C3b/iC3b (false color green) and endothelial marker vWF (false color red). The merge image indicates colocalization (white arrows) in orange. (C–E) Evaluation of the renal injury in WT and C3–/– mice (n = between 4 and 12 mice per group) injected with PBS or PHZ by analyzing gene expression by RTqPCR. (C) NGAL. (D) Kim-1. (E) HO-1. (F) Comparison of the quantification of the C3 fragment staining in glomeruli of mice injected with PBS, PHZ, heme, or hemoglobin (Hb) (n ≥ 10 mice per group). (G) C3b/iC3b (false green color) staining and quantification in glomeruli of frozen kidney sections of mice injected with PBS or PHZ, with or without hemopexin (Hx) at 24 hours after treatment. (H) C3b/iC3b (false green color) staining and quantification in glomeruli of frozen kidney sections of mice injected with PBS or PHZ, with or without haptoglobin (Hp) at 24 hours after treatment (n ≥ 4 mice per group). (I and J) C3b/iC3b staining (false color green) of kidney sections of mice injected with heme (I) or Hb (J), with or without Hx. Quantification of C3 staining in glomeruli (n ≥ 4 mice per group). * P < 0.05, ** P < 0.005, Mann-Whitney test (A, I, and J); Kruskal-Wallis with Dunn’s test for multiple pairwise comparisons (F–H); 2-way ANOVA with Tukey’s test for multiple comparisons (C–E). Values are shown as box plots with median and minimum/maximum points. Scale bar: 50 μm.

Article Snippet: Complement deposition was studied using rat anti-mouse C3 fragments (C3bi/C3b/C3c) IgG1 (Hycult Biotech, HM1065), visualized with goat anti-rat-AF488 (Invitrogen, A-11006) and rabbit anti-mouse C5b-9 (Abcam, ab55811), and visualized with goat anti-rabbit IgG-AF488 (Invitrogen, A-11008).

Techniques: Staining, Injection, Double Staining, Marker, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A) NHS was incubated with 50 μM hemin, 12.5 μM Hb (1 molecule of Hb contains 4 heme molecules) diluted in TBS. After a 30-minute incubation time at 37°C, the level of released Ba was measured by ELISA. *P < 0.005, Kruskal-Wallis with Dunn’s test for multiple pairwise comparisons. (B) HUVECs were treated with increasing concentrations of hemin or diluted in FCS-free culture medium (M199) for 30 minutes at 37°C. After removing the supernatant, HUVECs were exposed to NHS (33% final concentration diluted in the same medium) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition by flow cytometry. (C–F) HUVECs were treated with increased concentrations of hemin with or without 5 μM of Hx for 30 minutes at 37°C. After removing the supernatant, HUVECs were exposed to NHS (33% final concentration diluted in M199 FCS-free) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition (C and D) or C5b-9 formation (E and F) by flow cytometry. (C and E) Representative flow cytometry histograms. (D and F) Quantitative analyses. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, 2-way ANOVA with Sidak’s test for multiple comparisons. Values are shown as box plots with median and minimum/maximum points.

Article Snippet: Complement deposition was studied using rat anti-mouse C3 fragments (C3bi/C3b/C3c) IgG1 (Hycult Biotech, HM1065), visualized with goat anti-rat-AF488 (Invitrogen, A-11006) and rabbit anti-mouse C5b-9 (Abcam, ab55811), and visualized with goat anti-rabbit IgG-AF488 (Invitrogen, A-11008).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Flow Cytometry

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: (A and B) NHS was incubated with 1,000 MV/μl RBC MVs from HDs (n = 12) or SCD patients (n = 12) diluted in TBS. After 30 minutes at 37°C, the levels of released Ba (A) and sC5b-9 (B) were measured by ELISA. **P < 0.005, Mann-Whitney test. (C) Linear correlation between measured Ba and sC5b-9 levels with RBC MPs from SCD (n = 12) in tested samples. R2 = 0.4811; P = 0.0085. (D and E) NHS was incubated with 1,000 MV/μl MVs from SCD patients (n = 12) diluted in TBS with or without 25 μM Hx. After 30 minutes at 37°C, the levels of released Ba (D) and sC5b-9 (E) were measured by ELISA. *P < 0.05, ***P < 0.001, Wilcoxon test after a Shapiro-Wilk test for normality. (F and G) HUVECs were treated with 1,000 MV/μl RBC MPs from HDs (n = 4) or SCD patient (n = 7) diluted in M199 medium for 30 minutes at 37°C. Without removing the supernatant, HUVECs were exposed to NHS (final concentration at 33% diluted in M199 FCS-free) for 30 minutes at 37°C. Cells were detached and stained for C3 deposition by flow cytometry. (F) Flow cytometry histograms. (G) Quantitative analyses. *P < 0.05, Mann-Whitney test. (H) HUVECs were treated with 1,000 MV/μl RBC MPs from 1 SCD patient with or without 5 μM Hx. As above, NHS was added and cells were stained for C3 deposition by flow cytometry. Values are shown as box plots with median and minimum/maximum points.

Article Snippet: Complement deposition was studied using rat anti-mouse C3 fragments (C3bi/C3b/C3c) IgG1 (Hycult Biotech, HM1065), visualized with goat anti-rat-AF488 (Invitrogen, A-11006) and rabbit anti-mouse C5b-9 (Abcam, ab55811), and visualized with goat anti-rabbit IgG-AF488 (Invitrogen, A-11008).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Concentration Assay, Staining, Flow Cytometry

Journal: JCI Insight

Article Title: Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles

doi: 10.1172/jci.insight.96910

Figure Lengend Snippet: Extracellular heme can serve as an enhancer of the pathological process in SCD and other hemolytic diseases. (i) Heme activates EC by its proinflammatory and prooxidant effects. (ii) Heme induces hydrolysis of C3 into C3(H2O) and provokes direct complement activation in the serum. (iii) C3b generated by the fluid-phase C3 convertase will bind to the EC membrane and will form new AP C3 convertases that can locally autoamplify and generate C3b and C5b-9 deposition. (iv) Released C3a and C5a can bind to C3aR and C5aR to mediate proinflammatory processes. In the context of SCD, sickle RBCs will generate more heme-positive MVs. They carry heme, which will activate complement in serum and also on the EC surface. We hypothesize that this complement activation will contribute to the vascular injury and tissue damage in SCD. Hx was able to block, partially or completely, the hemolysis-induced complement activation in vitro and in vivo.

Article Snippet: Complement deposition was studied using rat anti-mouse C3 fragments (C3bi/C3b/C3c) IgG1 (Hycult Biotech, HM1065), visualized with goat anti-rat-AF488 (Invitrogen, A-11006) and rabbit anti-mouse C5b-9 (Abcam, ab55811), and visualized with goat anti-rabbit IgG-AF488 (Invitrogen, A-11008).

Techniques: Activation Assay, Generated, Blocking Assay, In Vitro, In Vivo