Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and development of an aptamer targeting C-type lectin-like molecule-1 as a biomarker for acute myeloid leukemia: a SELEX approach

doi: 10.3389/fbioe.2025.1601453

Figure Lengend Snippet: (A) Flow cytometry data for BSA with aptamer-2. (B) Flow cytometry data for the library. (C) Flow cytometry data for the library minus BSA. (D) Flow cytometry data for CLL-1 with aptamer-2. (E) The cumulative data of the scatter plot in the histogram above. (F) Response curve from the binding assay of aptamer-2 and CLL_1, showing the response (RU) over time (s) for different concentrations of the ligand (aptamer-2). It includes a dissociation constant (K d ) value, indicating the binding affinity of the aptamer.

Article Snippet: • Meanwhile, prepare 1 mg/mL of the target protein by dissolving recombinant CLL-1 (MedChemExpress) in 40 μL 0.1 M sodium acetate (NaAc) buffer, pH 5.5. (For negative controls, prepare 1 mg/mL bovine serum albumin (BSA) similarly.)

Techniques: Flow Cytometry, Binding Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Design and development of an aptamer targeting C-type lectin-like molecule-1 as a biomarker for acute myeloid leukemia: a SELEX approach

doi: 10.3389/fbioe.2025.1601453

Figure Lengend Snippet: (A) Superimposed docking results showing the top four binding poses of aptamer-2 with CLL-1. Chain A (green) and chain B (orange) of CLL-1 are depicted, illustrating potential binding variations. (B) A close-up view of the interaction interface between aptamer-2 and CLL-1, highlighting key molecular contacts. (C) Surface representation of CLL-1 (cyan) in complex with the top-ranked aptamer-2 binding conformation, revealing the structural fit. (D) Interaction map showing the surface regions of CLL-1 (cyan) that engage with aptamer-2 (orange), with interacting residues highlighted in magenta.

Article Snippet: • Meanwhile, prepare 1 mg/mL of the target protein by dissolving recombinant CLL-1 (MedChemExpress) in 40 μL 0.1 M sodium acetate (NaAc) buffer, pH 5.5. (For negative controls, prepare 1 mg/mL bovine serum albumin (BSA) similarly.)

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP gating strategy and in vitro results. (A) Transmission electron microscopy images of EP. (B) The EP gate was defined using 0.5 µm and 1.53 µm calibration beads. EP population shift was observed after staining with ASGR1 antibodies. (C) In vitro , TNF-α induced human hepatocyte–derived EP. Primary human hepatocytes following liver resection were isolated, cultured overnight, and stimulated with 10 and 20 ng/ml TNF-α. EP surface antigens were stained, and absolute AnnV + , CD130 + , Cx43 + , ASGR1 + EP were analyzed. The ordinary one-way ANOVA with Tukey’s post hoc test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Staining, Derivative Assay, Isolation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Time course of EP after LT (n = 11). (A) Total EP (/µl) (B) AnnV + , CD130 + , CD31 + EP (/µl) (C) Cx43 + , ASGR1 + , MDR3 + EP (/µl) were stained and analyzed. The Wilcoxon matched-pairs signed ranked test was used. The plots are indicated by the median, and all error bars indicate the interquartile range (IQR). A single asterisk indicates significance at p < 0.05 (D) Patients at risk for acute rejection. EP dynamics from preoperative state to POD 1 of the total, AnnV + , CD130 + , Cx43 + , ASGR1 + , MDR3 + , CD31 + EP (/µl) and their fold change were analyzed. Absolute EP were non-normally distributed; the two-tailed Mann-Whitney U test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP profiles during histology-proven ACR. EP were determined in the blood plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. EP surface antigens (A) AnnV + , (B) CD130 + , (C) Cx43 + , (D) ASGR1 + , (E) MDR3 + , (F) CD31 + EP were stained, and relative EP (%) were analyzed. (G) Example FSC-A (forward scatter) histograms of MDR3 + EP (%) (H) demonstrating the highest receiver operating characteristic (ROC) of all single antigens. One-way analysis of variance followed by Tukey’s post hoc test was used. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Staining

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Clustering and color coding with FlowSOM and viSNE for identifying EP subpopulations via density plots. (A) Qualitative analysis and population identification were performed using viSNE and FlowSOM on samples of patient 52, who had episodes of graft dysfunction caused by ACR and non-ACR. Several LBs were performed over time, and their concomitant blood samples presented the following data. t-SNE (t-distributed stochastic neighbor embedding), an unsupervised nonlinear dimensionality reduction algorithm, was used to fit six-dimensional data into two dimensions. All clusters were created via FlowSOM analysis. Clusters were formed based on FACS channels. The arrow indicates the novel ASGR1 + CD130 + AnnV + EP population. Coordinates for each t-SNE dimension (t-SNE1 and t-SNE2) were calculated for each microparticle after dimensionality reduction. (B) Color-coded t-SNE density plots showing different antigen expression levels by the channel of above-mentioned samples.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Analysis of ASGR1 + CD130 + AnnV + EP (%) population discovered by viSNE maps and FlowSOM algorithm. (A) The particular EP subpopulation was identified in the plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. ROC was constructed by comparing (B) ACR vs non-ACR and (C) ACR vs control. One-way analysis of variance followed by Tukey’s post hoc test was used. ROC curves and AUC were generated for relative EP. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Construct, Control, Generated

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Diagnosis sensitivity and specificity of ASGR1 + Annexin V + CD130 + EP (%) for ACR in liver transplant.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Biomarker Discovery

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

Journal: International Journal of Molecular Sciences

Article Title: Fraxin Attenuates Rheumatoid Arthritis by Regulating Macrophage Polarization and Inhibiting Fibroblast-like Synoviocyte Proliferation

doi: 10.3390/ijms27072946

Figure Lengend Snippet: Fraxin inhibited inflammation in Raw264.7 cells and FLs. ( A , B ) Effect of Fraxin on the cellular viability of Raw264.7 and FLs. ( C ) EC-50 of Fraxin (100 ng/mL) in Raw264.7 cells. ( D – F ) Flow cytometry analysis showing the polarization effect of Fraxin (100 ng/mL) on Raw264.7 cells. ( G – L ) RT-PCR analysis of Il6 , Tnfα , Il1b , Il10 , Cd206 , and Hspa8 in FLs treated with Fraxin. ( M – O ) Protein levels of p-NFκB-p65 and TNF-α were examined by Western blot in FLs. Fraxin low-dose group (Fraxin-L, 100 ng/mL), Fraxin high-dose group (Fraxin-H, 1 μg/mL). ## p < 0.01, ### p < 0.001 versus Ctrl; ** p < 0.01, *** p < 0.01 versus LPS.

Article Snippet: CD206 (A02285-2) and CD86 (BM4121) were purchased from Boster Biological Technology Co., LTD (Wuhan, China).

Techniques: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Fraxin Attenuates Rheumatoid Arthritis by Regulating Macrophage Polarization and Inhibiting Fibroblast-like Synoviocyte Proliferation

doi: 10.3390/ijms27072946

Figure Lengend Snippet: Fraxin improved the inflammatory microenvironment of the knee joint. ( A ) H&E staining of the knee joint. The area within the box represents the magnified region shown below. The red arrow points to meniscal injury, the blue arrow points to synovial thickening, and the green arrow points to synovial inflammatory pannus. ( B ) Knee joint staining with saffron solid green. ( C , D ) CD206 and CD86 immunofluorescence staining in the synovium. ( E ) The pathology score from H&E staining. ( F , G ) Quantitative analysis of CD206 and CD86 immunofluorescence staining. ( H ) Serum levels of IL-6 were analyzed by ELISA. ( I ) Serum levels of TNF-α were analyzed by ELISA. ## p < 0.01 versus Ctrl; ** p < 0.01 versus Model.

Article Snippet: CD206 (A02285-2) and CD86 (BM4121) were purchased from Boster Biological Technology Co., LTD (Wuhan, China).

Techniques: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: C-type lectin receptor 2d forms homodimers and heterodimers with TLR2 to negatively regulate IRF5-mediated antifungal immunity

doi: 10.1038/s41467-023-42216-3

Figure Lengend Snippet: a Flow cytometry assay of the competitive binding of C. albicans yeast cells with CLEC2D-hIgG1-Fc or hIgG1-Fc (hFc) using the indicated polysaccharides. b Representative immunofluorescent staining assay of the binding of CLEC2D-hIgG1-Fc (green) or Dectin-1-hIgG1-Fc (green) with C. albicans . Chitin was stained with Calcofluor White (50 μg/ml, blue), and β-glucan was stained with anti β-1,3-glucan antibody (red). Scar bars = 2 μm. Three times experiments were repeated independently with similar results. c ELISA assay for the binding of indicated amino acid point mutated proteins of CLEC2D-hIgG1-Fc or hIgG1-Fc (hFc) with plate-coated curdlan or α-mannan (5 μg/well). d ELISA results for the binding of diluted CLEC2D-Fc and TLR2-His homo- or heterodimers with plated-coated curdlan, α-mannan or Pam3CSK4 (1 μg/well), which were detected with anti-Fc or -His Abs, respectively. e ELISA results for the binding of CLEC2D-Fc/TLR2-His heterodimers containing the dilution of either CLEC2D-Fc (Red line) or TLR2-His (Black line) with plated-coated curdlan, α-mannan or Pam3CSK4 (1 μg/well), which were detected with anti-Fc or -His Abs, respectively. f Flow cytometry assay of the competitive binding of Dectin-1-hIgG1-Fc with the CLEC2D-Fc/TLR2-His heterodimers to C. albicans yeast cells, which was detected with anti-His Ab. Data were presented as mean ± SEM; n = 3 ( d – f ), n = 4 ( c ) biologically independent samples. Data were analyzed by unpaired two-sided Student’s t -test in ( c – f ). Source data are provided as a Source Data file.

Article Snippet: Anti-β-glucan antibody (Abcam, ab233743, dilution 1:100) and Dectin-1-Fc (MedChemExpress, HY-P70101, 10 μg/mL) or CLEC2D-Fc (10 μg/mL) proteins were used to bind with live C. albicans yeast cells.

Techniques: Flow Cytometry, Binding Assay, Staining, Enzyme-linked Immunosorbent Assay