sa00003  (Proteintech)

Journal: PLOS Pathogens

Article Title: Salmonella infection and biofilms drive anti-C1q and anti-dsDNA autoantibodies in systemic autoimmunity

doi: 10.1371/journal.ppat.1014666

Figure Lengend Snippet: ( A-B ) Mouse serum was assayed using a multiplex Luminex platform and antibodies quantified by mean fluorescence intensity (MFI); only anti-C1q antibodies are shown here, see for other antigens. 6-8 week old ( A ) C57BL/6 mice and ( B ) NZBxW/F1 mice were injected intraperitoneally with curli purified from Salmonella Typhimurium (STm) biofilms, or sterile PBS for controls, weekly for ( A ) 12 weeks or ( B ) 5 weeks. ( C-F ) Anti-C1q antibodies in serum (diluted 1:50) were detected using ELISA, normalized to aliquots of serum from an untreated aged MRL/lpr mouse as a positive control (1:50, top dotted line) and a negative control without serum (bottom dotted line). ( C ) C57BL/6 mice were injected intraperitoneally with 100 µg curli purified from STm biofilms, 100 µg/kg LPS, 50 µg curli and 50 µg/kg LPS, or PBS for untreated controls, weekly for 3 weeks . ( D ) C57BL/6 mice were injected intraperitoneally with 100 µg curli purified from the biofilm of an E. coli isolate from the human microbiome (hEC), an E. coli isolate from the mouse microbiome (mEC), STm biofilms, or PBS for untreated controls, weekly for 4 weeks . ( E ) 129Sv mice were infected intragastrically with 10 7 CFU of wild-type (WT) STm strain IR715, the isogenic curli-deficient mutant csgBA , or LB broth for uninfected controls, in two replicate experiments, and Week 6 serum was assayed. ( F ) C57BL/6 mice were infected intragastrically with 10 7 CFU of STm spiB or invAspiB mutant, or LB broth for uninfected controls, and monitored for 10 weeks. Week -1 denotes baseline samples collected from each group one week before the first injection (Week 0). Statistics: ( A-B ) Unpaired t-test, ( C-E ) One-way ANOVA, or ( E ) Mixed-effects analysis, with Tukey’s multiple comparisons, comparisons within the legend indicating overall column factor (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: Tissue was incubated in antibody cocktails including 1:30 goat anti-mouse IgG conjugated to AlexaFluor 405 (Invitrogen A31553), 1:25 rat anti-mouse C3 (11H9) conjugated to AlexaFluor 647 (Santa Cruz Biotechnology sc-58926), and 1:20 rat anti-mouse C1q (7H8) conjugated to FITC (Santa Cruz Biotechnology sc-58920) diluted in PBS-T with 1% bovine serum albumin for 2 hours at room temperature, covered with a small circle of parafilm and kept in the dark underneath a lid to prevent evaporation and bleaching.

Techniques: Multiplex Assay, Luminex, Fluorescence, Injection, Purification, Sterility, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Microbiome, Infection, Mutagenesis, Analysis

Journal: PLOS Pathogens

Article Title: Salmonella infection and biofilms drive anti-C1q and anti-dsDNA autoantibodies in systemic autoimmunity

doi: 10.1371/journal.ppat.1014666

Figure Lengend Snippet: Serum was collected from ( A, D ) pre-onset, ( B, E ) early-disease, and ( C,F ) late-disease NZBxW/F1 mice 24 hours after each weekly injection of curli purified from Salmonella Typhimurium biofilms or PBS for untreated controls. ( A-C ) ELISA assays detected anti-dsDNA antibodies in serum diluted ( A, B ) 1:250 or ( C ) 1:500 ( A-B, N = 8; C, N = 15). Week -1 denotes baseline samples collected from each group one week before the first injection (Week 0). ( D-F ) ELISA assays detected anti-C1q antibodies in serum diluted 1:50, normalized to the O. D. of positive control MRL/lpr serum, indicated by the top dotted line, and negative controls without serum are indicated by the bottom dotted line ( D-E, pre-bleed and Week 4 not assayed due to insufficient serum volume for the assay). Statistics: ( A-B, D-E ) Two-way ANOVA, or ( C, F ) Mixed-effects model, with Tukey’s multiple comparisons, comparisons within legends indicating overall column factor, (*p < 0.05, **p < 0.01).

Article Snippet: Tissue was incubated in antibody cocktails including 1:30 goat anti-mouse IgG conjugated to AlexaFluor 405 (Invitrogen A31553), 1:25 rat anti-mouse C3 (11H9) conjugated to AlexaFluor 647 (Santa Cruz Biotechnology sc-58926), and 1:20 rat anti-mouse C1q (7H8) conjugated to FITC (Santa Cruz Biotechnology sc-58920) diluted in PBS-T with 1% bovine serum albumin for 2 hours at room temperature, covered with a small circle of parafilm and kept in the dark underneath a lid to prevent evaporation and bleaching.

Techniques: Injection, Purification, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: PLOS Pathogens

Article Title: Salmonella infection and biofilms drive anti-C1q and anti-dsDNA autoantibodies in systemic autoimmunity

doi: 10.1371/journal.ppat.1014666

Figure Lengend Snippet: ( A ) Experimental scheme: MRL/lpr mice at age 6 weeks underwent weekly intraperitoneal injections of curli, purified from Salmonella Typhimurium biofilms or PBS for untreated controls, and periodic serum samples, taken 24 hours after each injection, for 6 weeks. ( B-C ) ELISA assays detected anti-dsDNA antibodies in serum diluted ( B ) 1:250 or ( C ) 1:500. ( D ) ELISA assay detected anti-C1q antibodies in serum diluted 1:50, normalized to the O.D. of the positive control untreated MRL/lpr serum. Dotted lines indicate relative O.D. of MRL/lpr positive control serum (top line) or negative control with no serum added (bottom line). Week -1 denotes baseline samples collected from each group one week before the first injection (Week 0). Statistics: Two-way ANOVA with Sidak’s multiple comparisons, comparisons within legends indicating overall column factor, (*p < 0.05, **p < 0.01).

Article Snippet: Tissue was incubated in antibody cocktails including 1:30 goat anti-mouse IgG conjugated to AlexaFluor 405 (Invitrogen A31553), 1:25 rat anti-mouse C3 (11H9) conjugated to AlexaFluor 647 (Santa Cruz Biotechnology sc-58926), and 1:20 rat anti-mouse C1q (7H8) conjugated to FITC (Santa Cruz Biotechnology sc-58920) diluted in PBS-T with 1% bovine serum albumin for 2 hours at room temperature, covered with a small circle of parafilm and kept in the dark underneath a lid to prevent evaporation and bleaching.

Techniques: Purification, Blood/Plasma/Serum Samples, Injection, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

Journal: PLOS Pathogens

Article Title: Salmonella infection and biofilms drive anti-C1q and anti-dsDNA autoantibodies in systemic autoimmunity

doi: 10.1371/journal.ppat.1014666

Figure Lengend Snippet: MRL/lpr mice were intraperitoneally injected weekly with ( A ) PBS or ( B ) curli purified from Salmonella Typhimurium for 6 weeks and euthanized at 13 weeks old. Frozen tissue sections were stained with goat anti-mouse IgG-AlexaFluor405 (blue), rat anti-mouse C3-AlexaFluor647 (red), and rat anti-mouse C1q-FITC (green). ( A-B ) Representative images of glomeruli by confocal microscopy (63X objective). Sections shown are from two individual mice per group, scale bars represent 20 µm. ( C-F ) Images were quantified in ImageJ for mean fluorescence intensity (MFI) inside glomeruli of ( C ) IgG, ( D ) C3, ( E ) C1q, and ( F ) area was measured, within at least 15 glomeruli per mouse and averaged. Statistics: ( C-F ) Unpaired t-test (**p < 0.01).

Article Snippet: Tissue was incubated in antibody cocktails including 1:30 goat anti-mouse IgG conjugated to AlexaFluor 405 (Invitrogen A31553), 1:25 rat anti-mouse C3 (11H9) conjugated to AlexaFluor 647 (Santa Cruz Biotechnology sc-58926), and 1:20 rat anti-mouse C1q (7H8) conjugated to FITC (Santa Cruz Biotechnology sc-58920) diluted in PBS-T with 1% bovine serum albumin for 2 hours at room temperature, covered with a small circle of parafilm and kept in the dark underneath a lid to prevent evaporation and bleaching.

Techniques: Injection, Purification, Tissue, Staining, Confocal, Microscopy, Fluorescence

Journal: Stem Cells International

Article Title: CD73‐Overexpressing ADSCs Promote Neurogenic Bladder Repair via A2A–JAK1/STAT3‐Mediated Modulation of Inflammation and Macrophage Polarization

doi: 10.1155/sci/8564160

Figure Lengend Snippet: CD73‐overexpressing ADSCs regulate macrophage polarization toward the M2 and inhibit NLRP3 inflammasome activation. (A) Flow cytometry results showing macrophage polarization in each group. CD86 was detected using FITC‐conjugated antibody, and CD163 was detected using PE‐conjugated antibody. (B) Western blot and quantitative analysis of NLRP3, ASC, and caspase‐1 expression in vitro. (C) Quantitative analysis of Western blot. (D) ELISA analysis of IL‐1β and IL‐18 levels in each group in vitro. Statistical significance is indicated as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. All experiments were independently performed in triplicate. The full‐length, uncropped Western blot images are provided in Figure . ELISA data are summarized in Tables and .

Article Snippet: For intracellular marker staining, cells were blocked with 5% bovine serum albumin and incubated for 30 min at room temperature with FITC‐conjugated antimouse CD86 (Elabscience, Wuhan, Hubei, China; 1:100 dilution) and PE‐conjugated antimouse CD163 antibodies (Elabscience, Wuhan, Hubei, China; 1:100 dilution).

Techniques: Activation Assay, Flow Cytometry, Western Blot, Analysis, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay