Journal: bioRxiv

Article Title: Antibodies against SARS-CoV-2 control complement-induced inflammatory responses to SARS-CoV-2

doi: 10.1101/2023.05.29.542735

Figure Lengend Snippet: (A-F) Human monocyte-derived DCs were exposed to SARS-CoV-2 isolate (hCoV-19/Italy-WT, 1000TCID/mL), complement-opsonized SARS-CoV-2 (hCoV-19/Italy-WT, 1000TCID/mL) and LPS (100 ng/mL) in presence or absence of anti-CD32 for 6 h. mRNA levels of APOBEC3G (A), IRF7 (B), CXCL10 (C), IL-6 (D), IL-10 (E) and IL-12p35 (F) were determine with qPCR after 6h (n=14 donors). Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A-F) 2-way ANOVA with Dunnett’s multiple-comparison test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (n=14 donors).

Article Snippet: The following antibodies were used (all anti-human): CD86 (2331 (FUN-1), BD Pharmingen), CD80 (L307.4, BD Pharmingen), PE-conjugated mouse IgG1 CR3/CD11b (101208, Biolegend), LEAF purified CR3/CD11b mouse IgG1, LEAF purified CR4/CD11c mouse IgG1, CR3/CD11b (M1/70), CR4/CD11c (S-HCL-3), CD32 (FUN-2), DC-SIGN (FAB161F, R&D systems), CD16, CD32 CD64 (all BD Pharmingen) and, viability dye (Ghost DyeTM Violet 510, Tonbo Biosciences, San Diego, USA).

Techniques: Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Antibodies against SARS-CoV-2 control complement-induced inflammatory responses to SARS-CoV-2

doi: 10.1101/2023.05.29.542735

Figure Lengend Snippet: (A-F) SARS-CoV-2 was pre-incubated with patient isolated mAb COVA1-18 and COVA1-27 (10µg/mL) for 30 min at 37°C. Human monocyte-derived DCs were exposed to SARS-CoV-2 isolate (hCoV-19/Italy-WT, 1000TCID/mL) alone or with blocks, to complement-opsonized SARS-CoV-2 (hCoV-19/Italy-WT, 1000TCID/mL) and LPS (100 ng/mL) in presence or absence of anti-CD32 for 2 h and 6 h. mRNA levels of IFNβ (A), APOBEC3G (B), IRF7 (C), CXCL10 (D), IL-6 (E) and IL-10 (F) were determined by qPCR (n=6 donors (A) and (n=4 donors) (B-F). Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A-F) 2-way ANOVA with Tukey’s multiple-comparison test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, (A) (n=6donors) and (B-F) (n=4 donors).

Article Snippet: The following antibodies were used (all anti-human): CD86 (2331 (FUN-1), BD Pharmingen), CD80 (L307.4, BD Pharmingen), PE-conjugated mouse IgG1 CR3/CD11b (101208, Biolegend), LEAF purified CR3/CD11b mouse IgG1, LEAF purified CR4/CD11c mouse IgG1, CR3/CD11b (M1/70), CR4/CD11c (S-HCL-3), CD32 (FUN-2), DC-SIGN (FAB161F, R&D systems), CD16, CD32 CD64 (all BD Pharmingen) and, viability dye (Ghost DyeTM Violet 510, Tonbo Biosciences, San Diego, USA).

Techniques: Incubation, Isolation, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Antibodies against SARS-CoV-2 control complement-induced inflammatory responses to SARS-CoV-2

doi: 10.1101/2023.05.29.542735

Figure Lengend Snippet: (A) SARS-CoV-2 pseudovirus opsonization patterns with mild and severe COVID-19 patient sera was determined by ELISA (p24 pg/mL) using anti-human C3c and C3d, for iC3b recognition, and anti-human IgG, for immunoglobulins detection. (B) Human monocyte-derived DCs were exposed to SARS-CoV-2 isolate (hCoV-19/Italy-WT, 1000TCID/mL), to complement-opsonized SARS-CoV-2 (hCoV-19/Italy-WT, 1000TCID/mL), COVID-19 patient serum (mild or severe) and antibody/complement-opsonized SARS-CoV-2 (hCoV-19/Italy-WT, 1000TCID/mL) in presence or absence of anti-CD32 for 2 h and 6 h. mRNA levels for IFNβ (B) were determined after 2 h and mRNA levels of IRF7 (C) and IL-6) (D) after 6 h by qPCR (n=6 donors) (B) and (n=8 donors) (C-D). Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B-D) ordinary one-way ANOVA with Tukey’s multiple-comparison test. *p ≤ 0.05, **p ≤ 0.01 (B) (n=6 donors) and (C-D) (n=8 donors).

Article Snippet: The following antibodies were used (all anti-human): CD86 (2331 (FUN-1), BD Pharmingen), CD80 (L307.4, BD Pharmingen), PE-conjugated mouse IgG1 CR3/CD11b (101208, Biolegend), LEAF purified CR3/CD11b mouse IgG1, LEAF purified CR4/CD11c mouse IgG1, CR3/CD11b (M1/70), CR4/CD11c (S-HCL-3), CD32 (FUN-2), DC-SIGN (FAB161F, R&D systems), CD16, CD32 CD64 (all BD Pharmingen) and, viability dye (Ghost DyeTM Violet 510, Tonbo Biosciences, San Diego, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Comparison

Journal: International journal of molecular sciences

Article Title: Analysis of CD1a-Positive Monocyte-Derived Cells in the Regional Lymph Nodes of Patients with Gallbladder Cancer.

doi: 10.3390/ijms252312763

Figure Lengend Snippet: Figure 2. Representative images of immunohistochemistry of CD209 (DC-SIGN) ((a) original mag- nification ×100, (b) original magnification ×200). (a) Many CD209-positive cells are found at the sinuses of LNs. (b) In metastatic LNs, CD209-positive cells are found at stroma surrounding cancer cells, some of them having dendritic shapes. (c) Image of immunohistochemistry of CD1a, almost the same area of (b). Dendritic-shaped CD1a-positive cells are found at stroma surrounding cancer cells (original magnification ×200).

Article Snippet: The following primary antibodies were used: the mouse monoclonal anti-CD1a antibody (clone 010; IS06930–2; prediluted; Dako, Glostrup, Denmark); the rabbit polyclonal antiS100 antibody (GA50461–2 J; prediluted; Dako); and the mouse monoclonal anti-CD209 antibody (clone 120507; R&D systems, Minneapolis, MN, USA).

Techniques: Immunohistochemistry

Journal: Respiratory research

Article Title: Differential proteins from EVs identification based on tandem mass tags analysis and effect of Treg-derived EVs on T-lymphocytes in COPD patients.

doi: 10.1186/s12931-024-02980-2

Figure Lengend Snippet: Fig. 5 Relative expressions of potential biomarkers. (A-E) The expression levels of (A) BTRC, (B) NCOA3, (C) TRIM28 ,(D) CD209, and (E) SSR3. (F) Protein bands. ▲p < 0.05, ▲▲p < 0.01 vs. healthy control

Article Snippet: The antibodies used in this experiment were listed as follow: BTRC Antibody (DF6534, Affinity; 1:1000); NCOA3 Antibody (AF4055, Affinity; 1:1000); TRIM28 Antibody (Bioss, bs-3581R; 1:1000); CD209 Antibody (DF2295 Affinity; 1:1000); SSR3 Antibody (DF2778, Affinity; 1:1000); Anti-rabbit IgG, HRP-linked Antibody (7074, CST; 1:6000); Vinculin Antibody (AF5122, Affinity; 1:1000); GAPDH Antibody (10494-1-AP, Proteintech; 1:10000).

Techniques: Expressing, Control

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Interaction of C1q, ghA, ghB, and ghC with DC-SIGN tetramer and monomer . (A) Microtiter wells coated with different concentrations (5, 2.5, 1.25, 0.625 µg/well) of DC-SIGN tetramer or monomer were probed with 2 µg/well of C1q. Bound C1q was detected with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP conjugate (1:1,000 in PBS). BSA was used as a negative control protein. (B) Binding of ghA, ghB, and ghC to DC-SIGN tetramer and (C) DC-SIGN monomer involved coating a range of concentrations of the respective proteins on microtiter wells, which were then incubated with a fixed concentration of ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl 2 buffer) at 37°C. Binding was detected using anti-MBP monoclonal antibodies (1:5,000 in PBS) and then rabbit anti-mouse IgG-HRP (1:5,000 in PBS). (D) Far western blot to show DC-SIGN tetramer binding to membrane-bound ghA, ghB, and ghC: 15 µg of ghA, ghB, and ghC (BSA and MBP as negative control proteins) were run on a 12% SDS-PAGE gel and then transferred on to nitrocellulose membrane. The blot was incubated with 50 µg of DC-SIGN in PBS overnight at room temperature. The bound DC-SIGN protein was detected using anti-DC-SIGN polyclonal antibodies and Protein A HRP conjugate. Bands were developed using diaminobenzidine tablets dissolved in water.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

Techniques: Negative Control, Binding Assay, Incubation, Concentration Assay, Bioprocessing, Far Western Blot, Membrane, SDS Page

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Interaction of C1q, ghA, ghB, and ghC with DC-SIGNR tetramer and monomer . (A) ELISA to examine binding of C1q to DC-SIGNR tetramer and SIGN-R monomer: DC-SIGNR tetramer or monomer were coated at different concentrations, followed by addition of 2 µg/well of C1q. Bound C1q was probed with anti-C1q polyclonal antibodies (1:1,000 in PBS) and Protein A HRP (1:1,000 in PBS), and the color was developed using o-phenylenediamine dihydrochloride. (B) Binding of ghA, ghB, and ghC to DC-SIGNR tetramer and (C) SIGN-R monomer: different concentrations of DC-SIGNR tetramer (B) and DC-SIGNR monomer (C) were coated on microtiter wells in carbonate buffer and incubated overnight at 4°C and then incubated with ghA, ghB, ghC, and MBP (2.5 µg/well in 5 mM CaCl 2 buffer). Binding was detected using anti-MBP monoclonal antibody and rabbit anti-mouse IgG-HRP conjugate.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: Competitive inhibition of DC-SIGN: HIV-1 gp120 interaction by globular head modules and gC1qR . (A) ELISA to assess whether gC1qR and ghB directly compete for the same binding site on DC-SIGN: DC-SIGN was coated at 5 µg/well overnight at 4°C. Wells were blocked with 2% BSA in PBS for 2 h at 37°C. gC1qR (5 µg/well) and different concentrations of ghB (5, 2.5, 1.25, 0.625 µg/well) were added in buffer containing 5 mM CaCl 2 . Incubation was carried out at 37°C for 1.5 h and 4°C for 1.5 h. Following repeated washes, bound gC1qR was probed using rabbit anti-gC1qR polyclonal antibodies (1:1,000) and Protein A-HRP (1:1,000). Color was developed using o -phenylenediamine dihydrochloride substrate; (B) competition between DC-SIGN tetramer and C1q globular head modules to bind solid-phase gp120. Microtiter wells were coated with 250 ng of gp120. Various concentrations of ghA, ghB, ghC, and C1q and constant 2.5 µg/mL of DC-SIGN were incubated at 37°C for 1 h and then at 4°C for 1 h. The binding of DC-SIGN to gp120 in the presence of globular heads or C1q was detected using rabbit anti-DC antibody (1:500), probed with Protein A HRP (1:5,000). DC-SIGN alone binding to gp120 was used as 100%.

Article Snippet: The blot was washed three times for 10 min each in PBS containing 0.05% Tween 20 and then incubated with anti-DC-SIGN (1:1,000) polyclonal antibody (ProSci) in 1% non-fat milk (2 h at 37°C).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation