hek blue il 6 reporter cells  (InvivoGen)

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Characterization of VHH clone diversity. ( A , B ) Dendrograms resulting from multiple sequence alignment of amino acid sequences of VHH clones specific for IL-6 ( A ) and IL-17A ( B ), performed using Clustal Omega [ https://www.ebi.ac.uk/jdispatcher/msa/clustalo ] (EMBL-EBI service providing bioinformatics tools; Accessed on 22 January 2026) . An asterisk (*) indicates clones that, at the phage library stage, contained a STOP codon in the coding sequence and required the presence of a corresponding suppressor tRNA provided by the E. coli host cell for protein synthesis. In red, two clones are marked that were selected at the end of the development stage for final BsAb assembly. ( C , D ) Residue conservation presented as a sequence logo representation of complementarity-determining regions’ (CDRs) repertoire among anti-IL-6 clones ( C ) and anti-IL-17A ones ( D ) obtained using WebLogo 3, a web-based application [ https://weblogo.threeplusone.com/create.cgi ] (accessed on 22 January 2026) [ , ]. The overall height of the stack at each position indicates the sequence conservation at that position, while the height of amino acid symbols within the stack represents the relative frequency of each amino acid. Moreover, the width of the stack is proportional to the fraction of the amino acid presence at a particular position, giving a richer picture of sequence conservation. The color scheme used shows amino acids according to their chemical properties.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Sequencing, Clone Assay, Residue

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Dissociation rates (k off in 1/s) of VHH clones specific for anti-IL-17A ( A ) and anti-IL-6 ( B ), were analyzed using bio-layer interferometry (BLI). The data illustrate the variability in dissociations rate across the selected clones for both IL-17A and IL-6. The clones highlighted in red, D3 for IL-17A and A5 for IL-6, represent the lead clones selected at a later stage of development.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Clone Assay

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: SDS-PAGE analysis of VHH clones under reducing conditions after purification using the KingFisher Flex station. Protein samples were loaded at 1 µg per well. Representative SDS-PAGE profiles of anti-IL-17A clones ( A ) and anti-IL-6 clones ( B ) illustrate the purity and integrity of the purified clones. The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: SDS Page, Clone Assay, Purification

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Purity analysis for all purified candidates from among anti-IL-17A clones ( A ) and anti-IL-6 clones ( B ) using capillary electrophoresis (LabChip system). The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red. Exemplary overlapped data analysis results obtained from the capillary electrophoresis are shown for anti-IL-17A clones ( C ) and anti-IL-6 clones ( D ).

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Purification, Clone Assay, Electrophoresis

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Melting temperature determination ( left panels ) for anti-IL-17A ( A ) and anti-IL-6 clones ( B ) performed using the Uncle platform, which enables the assessment of the thermal stability of the purified candidates. The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red. Representative melting curve analysis results for selected anti-IL-17A and anti-IL-6 clones are presented on the right panels .

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Clone Assay, Purification

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: The range of KD values for VHH clones specific to anti-IL-17A ( A ) and anti-IL-6 ( B ). The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red on the left panel . On the right panel , representative examples of BLI data analysis are shown. The red lines indicate the fitted curves, representing the global fitting of the binding data to a 1:1 interaction model.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Clone Assay, Binding Assay

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Neutralization potency of selected VHH clones targeting IL-17A ( A ) and IL-6 ( B ). The range of IC 50 values of VHHs is shown on the left panel . The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red. Right panel shows representative IC 50 determination analyses using reporter cell assays, where the dose-dependent inhibition of cytokine signaling was measured. Data were analyzed using nonlinear regression with a three-parameter dose–response model (log [inhibitor] vs. response), fitted using GraphPad Prism software. Each experiment was performed in two technical replicates.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Neutralization, Clone Assay, Inhibition, Software

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Relationship between IC 50 values from in vitro assays and binding affinity measured by BLI. ( A ) Correlation for IL-17A, with a correlation coefficient (R 2 ) of 0.8297. ( B ) Similar strong correlation for IL-6, as indicated by an R 2 of 0.7714. The most promising VHHs, D3 for IL-17A and A5 for IL-6, are marked in red.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: In Vitro, Binding Assay

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Binding affinity analysis of the CPBT0853 bispecific antibody. Representative bio-layer interferometry (BLI) binding curves for CPBT0853 interacting with human IL-17A ( A ), human IL-6 ( B ), human IL-17A/F heterodimer ( C ), cynomolgus monkey IL-17A ( D ), and cynomolgus monkey IL-6 ( E ) are shown. The absence of binding is demonstrated by the lack of interaction between CPBT0853 and mouse IL-6 ( F ).

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Binding Assay

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Blocking activity of the bispecific antibody CPBT0853 measured by bio-layer interferometry (BLI). Bar graphs represent quantitative data derived from BLI sensorgrams showing the effect of increasing concentrations of CPBT0853 on the binding of IL-17A ( A ) or IL-6 ( B ) to their respective receptors. The measured response [nm], reflecting the amount of receptor bound to the immobilized cytokine, decreases with increasing antibody concentration, indicating dose-dependent inhibition of cytokine–receptor interactions. Data represent mean ± SD from three independent experiments.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Blocking Assay, Activity Assay, Derivative Assay, Binding Assay, Concentration Assay, Inhibition

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Neutralization potency of CPBT0853 targeting IL-17A ( A ) and IL-6, IL-17A. Representative graphs show IC 50 analysis performed using a reporter cell assay. For comparison, parental VHHs and monospecific referential antibodies were used. The cellular response, measured as cytokine-induced activation, progressively declined with increasing concentrations of the tested antibodies, indicating effective inhibition of signaling through IL-6 ( A ), IL-17A ( B ), and the IL-17A/F heterodimer ( C ). Data were analyzed using nonlinear regression with a three-parameter dose–response model (log [inhibitor] vs. response), fitted using GraphPad Prism software. The curves shown represent a representative biological replicate performed with two technical replicates. The IC 50 values summarized in were calculated from three independent biological replicates.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Neutralization, Comparison, Activation Assay, Inhibition, Software

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Representative IC 50 curves showing inhibition of STATs phosphorylation in PBMCs stimulated with IL-6 and treated with increasing concentrations of the bispecific antibody CPBT0853. Phosphorylation of STAT1 ( A ) and STAT3 ( B ) was measured by flow cytometry. Data were normalized to cytokine-only controls and expressed as % pSTAT + lymphocytes. IC 50 values were calculated using a nonlinear regression with a three-parameter dose–response model (log [inhibitor] vs. response) in GraphPad Prism software. Each curve represents technical duplicates from PBMCs of one representative donor out of three tested.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Inhibition, Phospho-proteomics, Flow Cytometry, Software

Journal: Antibodies

Article Title: Development of Bispecific Antibody Targeting Human IL-17A and IL-6

doi: 10.3390/antib15020029

Figure Lengend Snippet: Inhibition of cytokine production (IL-6 and IL-8) in HFLS cells by CPBT0853. Levels of IL-6 ( A ) and IL-8 ( B ) secreted into the culture medium after stimulation of the cells with IL-17A. Additionally, cells were treated with bispecific and monospecific antibodies, as indicated. Data are shown as the mean ± SD from a minimum of four independent experiments. The bispecific antibody CPBT0853 effectively reduced the IL-6 and IL-8 levels, with higher efficiency, as compared to the monospecific antibodies, indicating enhanced anti-inflammatory activity. Additionally, the humanized IgG4 form of CPBT0853 (CPBT1269) was evaluated for comparison, showing activity in cytokine inhibition similar to that of the parental/original IgG1 form.

Article Snippet: HEK-Blue IL-6 reporter cells (#hkb-il6; InvivoGen) or HEK-Blue IL-17 reporter cells (#hkb-il17; InvivoGen) were used to assess the activity of the bispecific antibody CPBT0853, respectively.

Techniques: Inhibition, Activity Assay, Comparison

Journal: The EMBO Journal

Article Title: Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment

doi: 10.1038/s44318-025-00598-8

Figure Lengend Snippet: ( A ) Pull-downs with native and denatured (heat-inactivated and carbamidomethylated) C-terminally His-tagged C3-LHF1 were performed on human kidney homogenate. ( B ) Significantly enriched (BH-adj. t-test p -value < 0.05, log 2 FC > 1) proteins in the kidney pull-downs included the IL6 signaling component IL6ST (gp130). ( C ) Thermal proteome profiling by proteome integral solubility alteration assay (TPP-PISA) in human kidney lysates to identify putative C3-LHF1 interacting proteins ( n = 3 technical replicates). Only sign. altered (BH-adj. t-test p -value < 0.05, log 2 FC > 0.58) and plasma membrane-resident proteins are displayed after addition of 1 or 10 µM C3-LHF1 (1 h, 37 °C). Proteins stabilized by interactions with C3-LHF1 display a higher abundance in the treated samples (IL6ST depicted in red). ( D ) C3-LHF1 effect on IL6R/IL6ST signaling was tested in a HEK-Blue IL6Rα/IL6ST reporter line (incubation o/N at 37 °C). C3-LHF1 effect was concentration dependent, and activating, unlike bulk C3 ( n = 4, mean ± SE; two-sided t-test p -value < 0.001 = *; IL6 used at 3 × 10 −6 µg/mL, p -value vs. untreated control: 0.0000001; p -values for 5-10-20 µM C3-LHF1 vs. untreated control: 0.000119, 0.000009, and 0.000006, respectively). ( E ) In HEK-Blue IL6Rα/IL6ST cell line with both IL6 and C3-LHF1 (o/N at 37 °C), a partial competition can be observed at IL6 concentrations of >1 × 10 −4 µg/mL ( n = 4, mean ± SE). ( F ) Tocilizumab (TOC, 2 µg/mL for 3 h at 37 °C) did not inhibit the C3-LHF1 response in the IL6Rα/IL6ST cell line, unlike the IL6 response at 1 × 10 −4 µg/mL ( n = 4, mean ± SE, two-sided t-test p -value < 0.001 = *; p -value IL6 + tocilizumab vs. IL6 at 1 × 10 −4 µg/mL = 1.5 × 10 −9 ). .

Article Snippet: HEK-Blue IL6 reporter line , Invivogen , #hkb-hil6.

Techniques: Solubility, Clinical Proteomics, Membrane, Incubation, Concentration Assay, Control

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet: Response of IL6 trans -signaling by tissue-resident cells of articular joint (A and B) Flow cytometric histogram (A) and quantitative gMFI (B) showing the expression of respective receptors in primary mouse chondrocytes. n = 3 biological replicates. gMFI, geometric mean fluorescence intensity. (C and D) Experimental design (C) and heatmap (D) showing the secretion of interleukins, chemokines, and matrix enzymes from cartilage explants (n = 3) treated with hyper IL6, sgp130Fc, or their combination at the indicated doses. Each cell of heatmap represents the average expression value of cytokines from three technical replicates. Cytokines were measured using Luminex assay. (E) Representative TRAP stain images of BMMs (isolated from wild-type, il6st −/− , and tnsrsf11a −/− mice) treated with 50 ng/mL hyper IL6 or 100 ng/mL RANKL for 5 days. Scale bars, 500 μm. These micrographs are representative of three biological replicates for each group. (F) Surface area of TRAP-positive BMMs in (E) was measured using ImageJ software. Each dot represents the pixel size of an identified osteoclast. (G) Representative TRAP stain images of hyper IL6-induced BMMs treated with DMSO (<0.1%), 20 μg/mL AG490, or 5 μmol/L Stattic. Scale bars, 500 μm. These micrographs are representative of three biological replicates for each group. (H) Surface area of TRAP-positive BMMs in (G) was measured using ImageJ software. Each dot represents the pixel size of an identified osteoclast. The data are representative of two (A–D) or three (E–H) independent experiments with biologically independent samples. All data are presented as mean ± SEM. In (F) and (H), statistical significance was calculated using Kruskal-Wallis test with Dunn’s multiple comparisons test.

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Expressing, Fluorescence, Luminex, Staining, Isolation, Software

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet: Inflammatory stimulation promotes the release of IL6ST-bearing decoy EVs (A) EV quantification in blood and liver samples from DBA/1J (n = 3), K/BxN STA (n = 6), CIA (n = 6), or EAM mice (n = 3) using nanoparticle tracking analysis (NTA). (B and C) Protein lysates were collected from serum EVs from DBA/1J, K/BxN STA, CIA, and EAM mice. Representative western blot (B) and quantification analysis (C) of IL6ST are shown. β-tubulin was used as a loading control. n = 3 mice each group. (D) Representative transmission electron micrographs of serum EVs from indicated mouse models. Scale bars, 100 nm. (E) Total estimated number of IL6ST receptors per EV from serum of DBA/1J (n = 2), K/BxN STA (n = 6), CIA (n = 6), or EAM mice (n = 3) estimated based on obtained MFI values after fluorescence calibration of with Quantibrite polyethylene (PE) beads by flow cytometry. (F) Relative intensities of SEAP signal in HEK-Blue IL6 reporter cells induced by 10 ng/mL hyper IL6 and treated with 5 × 10 8 EV isolated from DBA/1J, K/BxN STA, CIA, or EAM mice. Data were normalized to HEK-Blue IL6 reporter cells treated with mock. Treatment with 200 ng/mL sgp130Fc was used as a positive control. n = 3 biological triplicates. Each dot represents the average value of three technical replicates. (G) Experimental design for EV transfer from K/BxN STA model mice to recipient CIA mice. (H and I) Clinical arthritis score over time (H) and at the endpoint (day 52) (I) in CIA mice treated with saline (n = 3), K/BxN STA mouse EVs (n = 5), or K/BxN STA mouse EVs preincubated with IL6ST mAb (n = 3) or IgG control (n = 3) recorded at the indicated time points. Each mouse was intravenously injected with 5 × 10 10 EVs. mAb, monoclonal antibody. The data are representative of two independent experiments with biologically independent samples (A–I). Data are presented as mean ± SEM. (A, C, E, F, and I) Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test for multiple comparisons.

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Western Blot, Control, Transmission Assay, Fluorescence, Flow Cytometry, Isolation, Positive Control, Saline, Injection

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet: Construction of engineered IL6ST decoy EVs by surface display of chimeric proteins (A) Schematic of DNA constructs expressing IL6ST-eGFP-CD63 and IL6ST-mCherry-syntenin. (B) Predicted accurate model building for the intracellular, extracellular, and transmembrane domains of IL6ST-eGFP-CD63 (pLDDT = 84.25), or IL6ST-syntenin (pLDDT = 81.22). A, Ig-like C2-type; B, fibronectin type-Ⅲ 1; C, fibronectin type-Ⅲ 2; D, fibronectin type-Ⅲ 3; E, fibronectin type-Ⅲ 4; F, fibronectin type-Ⅲ 5; pLDDT greater than 70 indicates that the predicted structure has a high degree of confidence. pLDDT, predicted local-distance difference test. (C) Schematic diagram showing two types of IL6ST decoy EVs obtained by fusing IL6ST to EV-sorting proteins in tandem with fluorescent proteins. (D) Relative intensities of SEAP signal in HEK-Blue IL6 reporter cells induced by 10 ng/mL of either hyper IL-6 (left) or IL6 (right) and treated with wild-type EVs, IL6ST-CD63 EVs, or IL6ST-syntenin EVs at the indicated doses. n = 3 biological replicates. Data were normalized to cells treated with wild-type EVs. (E) Protein lysates was collected from IL6ST decoy EVs or their donor cells. Western blot analysis was performed examining IL6ST, mCherry and eGFP. β-tubulin was used as a loading control. Specific bands corresponding to the predicted molecular weight of chimeric proteins are indicated by the black boxes. (F) Representative fluorescence images showing IL6ST decoy EVs labeled with eGFP (green) or mCherry (red) are internalized by HEK293 cells. Scale bars, 10 μm. (G) The number of eGFP or mCherry particles per cell in (F). The dot plot represents the number of eGFP or mCherry particles from individual cells. n = 20 cells each group. The data are representative of three independent experiments with biologically independent samples (D–G). Data are presented as mean ± SEM. (D) Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test. (G) Statistical significance was calculated using Kruskal-Wallis test with Dunn’s multiple comparisons test.

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Construct, Expressing, Western Blot, Control, Molecular Weight, Fluorescence, Labeling

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet: Co-expression of chimeric proteins optimizes the display of decoy receptors (A) Schematic diagram of the arrangement at the EV membrane of IL6ST fused to the sorting proteins in tandem with fluorescent proteins. (B) Representative transmission electron micrographs of decoy EVs with nanogold-labeled antibodies staining of IL6ST. Scale bars, 100 nm. Nanogold-labeled IgG antibodies was used as a negative control. These micrographs are representative of over 10 such images for each group. (C) Quantification of nanogold-labeled IL6ST epitopes at the EV membrane. The dot plot represents the number of nanogold particles from individual micrographs. n = 10 images each group. (D) Total estimated number of IL6ST receptors per EV estimated based on obtained MFI values after fluorescence calibration of with Quantibrite PE beads by flow cytometry. n = 3 biological replicates. (E) Heatmap showing the expression of interleukins, chemokines, and matrix enzymes in the supernatant of mouse cartilage explants treated with 50 ng/mL hyper IL6 and increasing doses of decoy EVs. Each cell of heatmap represents the average expression value of cytokines from three biological replicates. Cytokines were measured using Luminex assay. (F) Quantitative analysis showing the expression of six matrix enzymes in the supernatant of mouse cartilage explants treated with 50 ng/mL hyper IL6 and 3 × 10 9 /mL decoy EVs. Each dot represents the average expression value of cytokines from three biological replicates. Cytokines were measured using Luminex assay. (G) Representative TRAP stain images of BMMs treated with 50 ng/mL hyper IL6 and 5 × 10 9 decoy EVs for 5 days. Scale bars, 500 μm. These micrographs are representative of three biological replicates for each group. (H) Mature osteoclasts with three or more nuclei per well (n = 3) were counted. The data are representative of two (B–F) or three (G) independent experiments with biologically independent samples. All data are presented as mean ± SEM. (C and D) Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. (F) Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. (H) Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test for multiple comparisons.

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Expressing, Membrane, Transmission Assay, Labeling, Staining, Negative Control, Fluorescence, Flow Cytometry, Luminex

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet: Decoy EV treatment relieves disease phenotypes of several arthritis mouse models (A) Experimental design for assessing effects of decoy EV treatment on knee OA progression using a CIOA mouse model. (B) Clinical Osteoarthritis Research Society International (OARSI) score of disease progression in CIOA mice intraarticularly injected with saline (n = 3), 3 × 10 11 wild-type EVs (n = 3), 3 × 10 11 IL6ST decoy EVs (n = 6), or 3 × 10 11 combinatorial decoy EVs (n = 6) at the endpoint (day 42). The combinatorial EVs were composed of IL6ST, TNFR1, and IL1RⅡ decoy EVs in equal proportions (10 11 of each type). (C and D) Representative micro-CT images of sagittal views of subchondral bone medial compartment (C) and quantification of aberrant calcified tissue volume (Cal Tis. V) (D) in CIOA mice treated with saline (n = 3), 3 × 10 11 wild-type EVs (n = 3), 3 × 10 11 IL6ST decoy EVs (n = 6), or 3 × 10 11 combinatorial decoy EVs (n = 6) at the endpoint (day 42). Black arrows indicate osteophytes on the edge of the tibial plateau. (E) Experimental design for assessing the biodistribution of decoy EVs in diseased tissues of a CIA mouse model. 5 × 10 10 EVs were injected intravenously into mice in a single dose at day 10 or day 26, respectively. The distribution of decoy EVs in synovium and bone marrow was detected using NTA 6 h after EV injection. (F) EV numbers in synovium and bone marrow from sham mice (n = 3) or CIA mice (n = 6) were measured using NTA. (G) Experimental design for assessing the protective effects of decoy EVs on arthritis onset in a CIA mouse model. Mice were intravenously injected with saline, 2 mg/kg methotrexate, 3 × 10 11 wild-type EVs, or 3 × 10 11 combinatorial decoy EVs from day 3 to day 21 after the first immunization. (H) The incidence of arthritis in (H) was observed and recorded from day 21 to the endpoint (day 52). (I) Clinical arthritis score showing disease progression in CIA mice treated with saline (n = 3), 2 mg/kg methotrexate (n = 5), 3 × 10 11 wild-type EVs (n = 5), or 3 × 10 11 combinatorial decoy EVs (n = 12) over time. (J) Experimental design for assessing the therapeutic potential of decoy EVs on arthritis symptoms in a CIA mouse model. (K) Clinical arthritis score of disease progression in CIA mice treated with saline (n = 3), 3 × 10 10 combinatorial decoy EVs (n = 10), or 3 × 10 11 combinatorial decoy EVs (n = 10) over time. The combinatorial EVs were composed of IL6ST, TNFR1, and IL1RⅡ decoy EVs in equal proportions. (L) Heatmap and quantification of serum IL1β, TNFα, and IL6 in CIA mice treated with saline (n = 3), 3 × 10 10 decoy EVs (n = 10), or 3 × 10 11 decoy EVs (n = 10). Saline treatment was used as a negative control. Cytokines were measured using Luminex assay. (M) Schematic diagram showing proposed mechanism of action for therapeutic potential of decoy EVs in inflammatory arthritis. All data are presented as mean ± SEM. In (B) and (D), statistical significance was calculated using one-way ANOVA with Tukey’s post hoc test for multiple comparisons. In (F) and (L), statistical significance was calculated using Student’s two-sided t test. In (I) and (K), statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Biomarker Discovery, Injection, Saline, Micro-CT, Negative Control, Luminex

Journal: Cell Reports Medicine

Article Title: Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis

doi: 10.1016/j.xcrm.2023.101228

Figure Lengend Snippet:

Article Snippet: HEK-BlueTM IL6 Reporter cells , InvivoGen , hkb-hil6; RRID: CVCL_UF60.

Techniques: Recombinant, Adjuvant, Staining, BIA-KA, Luminex, Fluorescence, Quantitation Assay, RNA Expression, Software