Journal: Osteoarthritis and cartilage open

Article Title: Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model

doi: 10.1016/j.ocarto.2020.100099

Figure Lengend Snippet: Bovine articular cartilage explants (3 mm x 1 mm cylinders including the superficial zone, 4 per well) were cultured for three weeks according to the following treatment conditions: (N) untreated controls; (I) a single applied mechanical impact injury (50% final strain at a strain rate of 100%/s, followed by immediate release at the same rate); (C), addition of cytokines: TNFα, IL-6 and sIL-6R (10 ng/ml, 20 ng/ml, and 100 ng/ml, respectively) (IC), applied injury plus addition of cytokines; (D), untreated control + Dex (100 nM); (ID), applied injury + Dex; (CD), cytokines + Dex; or (ICD), applied injury + cytokines + Dex. Culture medium was changed every two days, and three biological replicates (animals) were used for all treatment conditions.

Article Snippet: After pre-equilibration, samples were treated for 22 days as in : no treatment (N), mechanical injury at day 0 (a single unconfined compression at 50% final strain, 100%/s strain rate, followed by immediate release at the same rate[ 5 ]; treatment I); cytokines (10 ng/ml recombinant human TNFα, 20 ng/ml recombinant human IL-6 and 100 ng/ml sIL-6R (R&D Systems); treatment C); injury+cytokines (treatment IC), with all four treatment groups additionally receiving 100 nM Dex (treatments D, ID, CD, and ICD, respectively).

Techniques: Cell Culture, Control

Journal: Osteoarthritis and cartilage open

Article Title: Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model

doi: 10.1016/j.ocarto.2020.100099

Figure Lengend Snippet: (A) A Venn diagram reveals a large overlap of MS-identified proteins between biological replicates. 500 proteins were found in medium samples from all three animals, and few proteins were found in only one or two animals. (B) Principle component analysis (PCA) was performed using abundance values for 456 filtered proteins obtained from MS analysis of cartilage explant medium samples taken on day 4, 8, 12, 16, and 20 of culture from the treatment groups control (N), injury (I), cytokines (C), and injury+cytokines (IC). The data clearly separate into two clusters, one with treatments N and I, and the second with treatments C and IC. Percentages on axes represent percent variance explained by that principal component. The large symbol within each cluster represents the cluster centroid.

Article Snippet: After pre-equilibration, samples were treated for 22 days as in : no treatment (N), mechanical injury at day 0 (a single unconfined compression at 50% final strain, 100%/s strain rate, followed by immediate release at the same rate[ 5 ]; treatment I); cytokines (10 ng/ml recombinant human TNFα, 20 ng/ml recombinant human IL-6 and 100 ng/ml sIL-6R (R&D Systems); treatment C); injury+cytokines (treatment IC), with all four treatment groups additionally receiving 100 nM Dex (treatments D, ID, CD, and ICD, respectively).

Techniques: Control

Journal: Osteoarthritis and cartilage open

Article Title: Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model

doi: 10.1016/j.ocarto.2020.100099

Figure Lengend Snippet: Treatment effects were evaluated by pairwise comparisons of MS abundance data (log2 summed ratios with imputation of missing values, FDR = 0.05) of different disease treatments within each animal replicate. Proteins were selected that had a differential effect of C or IC treatments and that were present in at least three time points across at least one consistent treatment condition between all three biological replicates (to avoid biases from imputing missing values), resulting in 188 selected proteins. The raw abundance value for each filtered protein was summed over all timepoints and log2-transformed. For visualization, the log2-transformed values were normalized via z-scoring across all treatment conditions, excluding injury alone and injury with Dex: control (N), cytokine (C), injury+cytokines (IC), Dex (D), cytokines+Dex (CD), and injury+cytokines+Dex (ICD). Proteins are plotted on the horizontal axis, and ordered based on their hierarchical clustering (Euclidian distance) across all six selected treatment conditions. Each individual replicate is plotted on the vertical axis, ordered by treatment condition and then by animal. The clustering reveals three major patterns of protein release: increased release by cytokines alone (↑Cyt), an increase by both cytokines and injury+cytokines (↑Cyt, ↑Inj+Cyt), and decreased release by cytokines and injury+cytokines (↓Cyt, ↓Inj+Cyt). These three patterns were used to designate the grouping categories in Table 1.

Article Snippet: After pre-equilibration, samples were treated for 22 days as in : no treatment (N), mechanical injury at day 0 (a single unconfined compression at 50% final strain, 100%/s strain rate, followed by immediate release at the same rate[ 5 ]; treatment I); cytokines (10 ng/ml recombinant human TNFα, 20 ng/ml recombinant human IL-6 and 100 ng/ml sIL-6R (R&D Systems); treatment C); injury+cytokines (treatment IC), with all four treatment groups additionally receiving 100 nM Dex (treatments D, ID, CD, and ICD, respectively).

Techniques: Transformation Assay, Control

Journal: Osteoarthritis and cartilage open

Article Title: Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model

doi: 10.1016/j.ocarto.2020.100099

Figure Lengend Snippet: The simplified graphical representation for each protein shows the ratio for each treatment comparisons: injury (I), cytokines (C), and injury+cytokines (IC) versus control (N), and treatments with Dex versus their non-Dex controls: D vs. N, ID vs. I, CD vs. C, and ICD vs. IC. The mean fold change values from three replicates are indicated with horizontal lines. Representative proteins for the different profile categories (Table 1): (A) Group I, collagen type II (COL2A1); (B) Group II, phosphoglycerate kinase (PGK1); (C) Group III, collagenase-3 (MMP-13). Also shown are (D) connective tissue growth factor (CTGF), a representative of proteins with a consistent effect of Dex after addition to any treatment, and (E) cartilage oligomeric matrix protein (COMP), which undergoes no statistically significant change in release with any treatment.

Article Snippet: After pre-equilibration, samples were treated for 22 days as in : no treatment (N), mechanical injury at day 0 (a single unconfined compression at 50% final strain, 100%/s strain rate, followed by immediate release at the same rate[ 5 ]; treatment I); cytokines (10 ng/ml recombinant human TNFα, 20 ng/ml recombinant human IL-6 and 100 ng/ml sIL-6R (R&D Systems); treatment C); injury+cytokines (treatment IC), with all four treatment groups additionally receiving 100 nM Dex (treatments D, ID, CD, and ICD, respectively).

Techniques: Control

Journal: Osteoarthritis and cartilage open

Article Title: Proteomic analysis reveals dexamethasone rescues matrix breakdown but not anabolic dysregulation in a cartilage injury model

doi: 10.1016/j.ocarto.2020.100099

Figure Lengend Snippet: (A) Cartilage explants (n=3) were cultured from 2 up to 22 days. The amounts of sGAG (mean ± SD) released at each day of culture was measured by the DMMB assay for all treatment groups: control (N), injury alone (I), cytokine (C), injury+cytokines (IC), Dex (D), injury+Dex (ID), cytokines+Dex (CD), and injury+cytokines+Dex (ICD). The release of sGAG was elevated with cytokine treatment; the addition of Dex reduced this release. (B) Medium samples were deglycosylated and run (44 to 100μl medium/lane) on 3–8% Tris-acetate SDS-gels and applied for Western blot using ARGS-aggrecan N-terminal neoepitope antibodies. Bovine protein fragments (previously described[50]) and their molecular weight in kDa are shown at the right. CS2 = chondroitin sulfate region 2. Cyt = cytokine treatment, Inj = applied injury, Dex = Dex treatment.

Article Snippet: After pre-equilibration, samples were treated for 22 days as in : no treatment (N), mechanical injury at day 0 (a single unconfined compression at 50% final strain, 100%/s strain rate, followed by immediate release at the same rate[ 5 ]; treatment I); cytokines (10 ng/ml recombinant human TNFα, 20 ng/ml recombinant human IL-6 and 100 ng/ml sIL-6R (R&D Systems); treatment C); injury+cytokines (treatment IC), with all four treatment groups additionally receiving 100 nM Dex (treatments D, ID, CD, and ICD, respectively).

Techniques: Cell Culture, Dimethylmethylene Blue Assay, Control, Western Blot, Molecular Weight

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: Ta-DCs secreted high levels of inflammatory mediators and promoted the invasion of tumor cells in vitro. (A) BMDCs were stimulated with PBS or 5 μg/ml of B16-F10-EXO, 3LL-EXO or 4T1-EXO for 6 h. Production of IL-6 and PGE2 from BMDCs was measured by ELISA (n = 3). (B) B16-F10, 3LL or 4T1 tumor cells were cultured in supernatant from corresponding exosome-stimulated BMDCs for 24 h, and the cells were plated in the top chamber of a Transwell plate. Twenty-four hours later, the cells on the bottom of the Transwell filter were imaged and quantified. (C) The results of (B) were statistically analyzed (n = 5). (D) B16-F10, 3LL or 4T1 tumor cells were cultured in supernatant from corresponding exosome-stimulated BMDCs for 24 h, and the cells were plated in the top chamber, precoated with 50 μl of Matrigel. Forty-eight hours later, the cells on the bottom of the Transwell filter were imaged and quantified. (E) The results of (D) were statistically analyzed (n = 5). (F) B16-F10, 3LL or 4T1 tumor cells were cultured in supernatant from corresponding exosome-stimulated BMDCs for 24 h, and the cells were then collected and re-cultured in fresh medium. The proliferation of tumor cells was measured using an alamarBlue assay at 24 h, 48 h and 72 h (n = 5). (A, C, E, F) The results are shown as the mean ± SEM of 3 independent experiments. (B, D) One representative image out of 5 is shown (Magnification: 200 ×). P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; ***p < 0.001.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture, Alamar Blue Assay, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: Activation of STAT3 by IL-6 from Ta-DCs increased transcription of MMP9. (A, B) B16-F10 cells were cultured in supernatant from PBS-stimulated BMDCs of WT mice or B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs of WT or IL-6−/− mice for the indicated time points. Then, the p-STAT3 and STAT3 protein levels were detected by Western blot (A), or with or without 2 μM of Stattic for 6 h. Then, the MMP9 mRNA level was detected by real-time PCR (B). (C-E) B16-F10 cells were cultured in supernatant from PBS- or B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs with or without 2 μM of Stattic for 24 h. Then, the MMP9 protein level was detected by Western blot (C), or the cells were collected and cultured in serum-free DMEM media for another 24 h. The MMP2 and MMP9 activity in the supernatant was detected by gelatin zymography (D), or the invasive ability of B16-F10 cells was measured using an in vitro invasive assay (n = 5) (E). (F) B16-F10 cells were stimulated with or without 10 ng/ml of IL-6 for 24 h, and the binding of STAT3 and the MMP9 promoter was examined by ChIP assay. (G) NIH-3T3 cells were transfected with MMP9-luc, along with STAT3 plasmids, with or without 10 ng/ml of IL-6 stimulation. Twenty-four hours later, luciferase activity was analyzed and normalized to that of Renilla luciferase. (A, C) Numbers indicate the ratio of gray values of the corresponding protein to that of STAT3 or β-Actin. (A, C, D) One representative of 3 independent experiments is shown. (B, E-H) The results are shown as the mean ± SEM of 3 independent experiments (n = 3). P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Activation Assay, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Zymography, In Vitro, Binding Assay, Transfection, Luciferase, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: IL-6 from Ta-DCs promoted tumor cell invasion through upregulation of MMP9 expression. (A-C) B16-F10 cells were cultured in supernatant from PBS- or B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs in the presence of 30 μg/ml of anti-IL-6, PGE2 or isotype control (ISO) mAbs (A), or BMDCs of WT or IL-6−/− mice (B) for 24 h. Their invasive ability was measured using an in vitro invasive assay (n = 5) (A, B). The MMP2, MMP9 and MMP13 protein levels in each supernatant-treated B16-F10 cells were detected by Western blot (C). (D, E) B16-F10 cells were cultured in supernatant from B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs from WT or IL-6−/− mice for 24 h, and the MMP9 protein level was detected by Western blot (D), or in the presence of 600 nm MMP9 inhibitor for 24 h. Then, the invasive ability of B16-F10 cells was measured using an in vitro invasive assay (n = 5) (E). (A, B, E) The results are shown as the mean ± SEM of 3 independent experiments. (C, D) One representative of 3 independent experiments is shown. Numbers indicate the ratio of gray values of the corresponding protein to that of β-Actin. P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; **p < 0.01; ***p < 0.001; NS, not significant.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Expressing, Cell Culture, In Vitro, Western Blot, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: HSP72 and HSP105 on the surface of TEXs mediated TLR2- and TLR4-dependent IL-6 secretion of DCs. (A) BMDCs were pre-incubated with anti-TLR2, TLR4 or both mAbs at a concentration of 30 μg/ml for 1 h, and BMDCs were stimulated with 5 μg/ml B16-F10-EXO for 6 h. IL-6 production from BMDCs was measured by ELISA. (B, C) Exosomes from B16-F10 cells transfected with HSP72, HSP105 or HSC70 siRNA, or NC siRNA were isolated. The HSP protein knockdown effect was detected by Western blot (B). BMDCs were stimulated with 5 μg/ml of the indicated B16-F10-EXO for 6 h, and IL-6 production from BMDCs was measured by ELISA (C). (D, E) After adsorption onto latex beads (D) or anti-CD63-coated latex beads (E), HSP72 and HSP105 on exosomes were detected by flow cytometry. (F, G) BMDCs were pretreated with 2.5 μg/ml cytochalasin D for 30 min, and co-cultured with CFSE-labeled B16-F10-EXO for 6 h. The uptake of B16-F10-EXO by BMDCs was detected by confocal microscopy (F), or stimulated with B16-F10-EXO for 6 h. IL-6 production by BMDCs was measured by ELISA (G). (B) Numbers indicate the ratio of gray values of the corresponding protein to that of CD63. (A, C, G) The results are shown as the mean ± SEM of 3 independent experiments (n = 3). (B, D, E, F) One representative of 3 independent experiments is shown. P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; *p < 0.05 and ***p< 0.001 versus B16-F10-EXO in A.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Isolation, Western Blot, Adsorption, Flow Cytometry, Cell Culture, Labeling, Confocal Microscopy, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: HSP105 acts as a ligand of TLR2 and TLR4 to induce DC IL-6 production. (A) Affinity precipitation of recombinant His-TLR2 or His-TLR4 protein with Flag-EGFP or Flag-HSP105 protein, captured by anti-FLAG® M2 magnetic beads and analyzed by immunoblot analysis with Abs against TLR2 and TLR4. (B) BMDCs were pre-incubated with anti-TLR2, TLR4 or both mAbs at a concentration of 30 μg/ml for 1 h, and then stimulated with 2 μg/ml endotoxin-free Flag-EGFP or Flag-HSP105 protein for 6 h. IL-6 production was measured by ELISA (n = 3). (A) One representative of 3 independent experiments is shown. (B) The results are shown as the mean ± SEM of 3 independent experiments. P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Affinity Precipitation, Recombinant, Magnetic Beads, Western Blot, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro. (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t-test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; ***p < 0.001.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: In Vitro, Adsorption, Flow Cytometry, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: HSP72 and HSP105 mediated Ta-DCs promotion of B16-F10 tumor lung metastasis via an IL-6/MMP9 pathway in vivo. (A, B) B16-F10 cells were cultured in supernatant from PBS-stimulated BMDCs of WT mice, or B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs of WT or IL-6−/− mice for 24 h. Then, 5 × 105 tumor cells were intravenously injected into mice, and these mice were killed 15 d later. Representative gross morphology of the lungs is shown (A). Numbers of invasive nodules in the lungs were counted and statistically analyzed (B). (C) The MMP9 protein level in B16-F10-MMP9(KD cells was detected by Western blot. (D, E) B16-F10-MMP9(KD) cells were cultured in supernatant from B16-F10-EXO (5 μg/ml for 6 h)-stimulated BMDCs for 24 h. Then, 5 × 105 tumor cells were intravenously injected into mice, and these mice were killed 15 d later. Representative gross morphology of the lungs is shown (D). Numbers of invasive nodules in the lungs were counted and statistically analyzed (E). (F, G) B16-F10 cells were cultured in supernatant from B16-F10-EXO/(EV)-, B16-F10-EXO/HSP72(OE)- or B16-F10-EXO/HSP105(OE) (5 μg/ml for 6 h)-stimulated BMDCs for 24 h. Then, 5 × 105 tumor cells were intravenously injected into mice, and these mice were killed 15 d later. Representative gross morphology of the lungs is shown (F). Numbers of invasive nodules in the lungs were counted and statistically analyzed (G). (C) Numbers indicate the ratio of gray values of MMP9 to that of β-Actin. (A, C, D, F) One representative of 3 independent experiments is shown. (B, E, G) The results are shown as the mean ± SEM of 3 independent experiments (n = 5). P values were generated by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test; **p < 0.01; ***p < 0.001. Control indicates B16-F10 cells without any treatment.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: In Vivo, Cell Culture, Injection, Western Blot, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: Depletion of IL-6 signaling converted TEXs from promoters to inhibitors of tumor metastasis in vivo. (A, B) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO labeled with or without PKH26 and killed 24 h later. Then, DCs and nuclei in the sections of spleens were stained with CD11c and DAPI, respectively. The binding of DCs and B16-F10-EXO was detected with an immunofluorescence assay. Arrows indicate the binding (A). DCs in splenocytes were isolated. The expression of MHC-II, CD80, CD86 and CD40 molecules on DCs were detected by flow cytometry (B). (C, D, E) Mice with an intravenous injection of 5 × 105 B16-F10 or B16-F10/OVA cells on day 0, were intravenously injected with 100 μl of PBS, 10 μg/100 μl of B16-F10-EXO or B16-F10/OVA-EXO on days 4, 7 and 10. Mice were killed and numbers of invasive nodules in the lungs were counted and statistically analyzed on day 15 (n = 5) (C). The OVA257–264 peptide specific CD8+ T cells in the lungs were detected by Tetramer assay (D). Data in (D) were statistically analyzed (n = 3) (E). (A, B, D) One representative of 3 (A, B) or 2 (D) independent experiments is shown. (C, E) The results are shown as the mean ± SEM of 3 independent experiments. P values were generated by a 2-tail student's t-test.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: In Vivo, Injection, Labeling, Staining, Binding Assay, Immunofluorescence, Isolation, Expressing, Flow Cytometry, Generated

Journal: Oncoimmunology

Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

doi: 10.1080/2162402X.2017.1362527

Figure Lengend Snippet: DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 105 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DCPBS or SN-DCtumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t-test.

Article Snippet: Recombinant mouse and human granulocyte/macrophage colony-stimulating factor (GM-CSF), mouse PGE2 ELISA kits, recombinant His-TLR2, His-TLR4/MD-2 complex, and mouse (MAB406) and human (MAB2061R) neutralizing IL-6 mAbs were purchased from R&D Systems.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro, Injection, Generated

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Initial Elispot Harmonization guidelines, as published in [4]

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Anatomy of a typical spot. After finishing the ELISpot assay developed with BCIP/NBT chromogen and immobilizing cells to the PVDF membrane, the mem- brane was removed from the plate and sectioned on the cryostat in a perpen- dicular direction. DAPI was used to counterstain the nuclei of immobilized cells (arrowhead). Spot boundaries indicate that cell-secreted proteins can migrate away from the cell in distances that exceed the size of the cell 3–5 times

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot, Membrane

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 5 Controls recommended for running side by side with experimental groups. Several controls are recom- mended to make sure that experimental conditions and reagents are not causing the formation of nonspecific spots: (1) Background control—complete ELISpot development with no cells added to wells; (2) No-detection antibody control—cells are added to the wells but ELISpot development omits adding detection antibodies; (3) Non-stimulated cells control—complete ELISpot development using non-stimulated cells (see Note 12); and (4) Immunoassay control—complete ELISpot development adding a corresponding recombinant protein rather than stimulated cells

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Control, Enzyme-linked Immunospot, Recombinant

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Effects of illumination conditions on the results of the automated search process. (a) Field of view in bottom of the ELISPOT well is evenly illuminated which results in almost a uniform background. (b) Illumination is uneven and as a result, the software easily detects sharp round spots, whereas blurry spots that have irregu- lar form may remain undetected

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A schematic flowchart of ELISpot assay to detect Ag-specific ASCs. (A) Pre-coat the wells of the ELISpot plate with an Ag, e.g., tetanus toxin. (B) Seed serial diluted PBMCs into wells of the plate, respectively. Culture overnight (minimum: 8 h). (C) Wash off cells with PBS-T. (D) Add AP-conjugated detection Abs specific to IgM, IgG, or IgA. (E) Wash off unbound Abs. (F) Develop the spots with BCIP/NBT substrate solution

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Flowchart detailing the ELISPOT protocol at a glance

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical cultured ELISpot response to the panel of DENV serotype specific peptides. (a) The layout of the ELISpot for the 17 DENV serotype specific peptides is shown, with each peptide response done in duplicate. (b) A response to these peptides from one dengue seropositive donor is shown with responses to DENV2 pep- tide 11 and 18, DENV4 peptide 5 and 19 and PHA. Therefore, as this individual responds to SS peptides of DENV2 and DENV4, it is likely that he/she has been infected with these two DENV serotypes in the past

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical ex vivo ELISpot assay with PHA as the positive control, DENV NS3 overlapping peptides and media as the negative control

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Ex Vivo, Enzyme-linked Immunospot, Positive Control, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Typical IFN-γ ELISpot images showing antigen-specific responses to serotypes of DENV E protein from a representative DENV-immune and a DENV-naïve subject

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs). The two MAPs on the left (top and bottom) consist of FIV p24 peptides (Fp9-3 and Fp14-3/4), and those on the right consist of FIV reverse transcriptase (RT) peptides (FRT3-3/4 and FRT7-1/2). The sequences of the long peptide and its smaller over- lapping peptides are shown for Fp14-3/4 (Fp14-3, Fp14-4), FRT3-3/4 (FRT3-3, FRT3-4), and FRT7-1/2 (FRT7- 1, FRT7-2). Four identical FIV peptides are on the amino-end of the MAP attached to a branched lysine backbone with palmitic acid (Pam) on the carboxyl-end. The final MAP vaccine consisted of all four MAPs at equal amounts (100 μg/dose each; total 400 μg/dose). Each MAP and its long and short peptides were used as FIV peptide stimulants in the IL2, IFNγ, and IL10 ELISpot assays and the T-cell proliferation analysis

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Reverse Transcription, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Influenza A/H1N1-specific memory B cell response pre- and post-influenza vaccination. Figure 1 demonstrates the dynamics of influenza-specific memory B cell ELISPOT response after influenza vaccination (in a cohort of 106 healthy older adults, 50–74 years old) and has been previously published in Viral Immunology [7] and Plos One [8]. The top (bottom) of the box indicates the 75th (25th) percentiles, respectively, while the bold line within the box indicates the median. The “whiskers” extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles, respectively. Beyond that point, individual points are plotted. B cell ELISPOT counts representing the influenza A/H1N1- specific IgG-producing memory-like B cell response plotted for each timepoint as spot forming units (SFUs) per 2 × 105 PBMCs

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Stem Cell Research & Therapy

Article Title: Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes

doi: 10.1186/scrt466

Figure Lengend Snippet: Quantification of collagens I, II, and III and glycosaminoglycan (GAG) in fibrin gel lysates. Protein synthesis of collagens (A) I, (B) II, (C) III, and (D) GAG in cell lysates after 28 days of culture. Bone marrow-derived stem cells (BMSCs) (white bars), mixed cultures (BMSCs and chondrocytes in a ratio of 1:1, light grey bars), or chondrocytes (dark grey bars) were kept as monocultures (F, bars with pattern) or as co- and tri-cultures with osteoarthritis cartilage explants (FC, blank bars) in chondrogenic medium. (A, B) Collagens I and II were quantified with enzyme-linked immunosorbent assay or (C) for collagen III by densitometrically evaluated dot-blot analysis including a recombinant collagen III standard curve. (D) GAG concentration in cell lysates was quantified by a dimethylmethylene blue assay including a chondroitin sulfate standard curve. Results are presented as mean with standard deviation. * P <0.05; ** P <0.01. N = 7. Ch, chondrocytes.

Article Snippet: To determine the concentration of specific proteins in the supernatant, the human IL-1β sandwich ELISA kit (RayBiotech), IL-6 sandwich ELISA kit (R&D Systems), and IL-8 ELISA Kit (Gen-Probe, now part of Hologic, Bedford, MA, USA) were used in accordance with the instructions of the manufacturers.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Dot Blot, Recombinant, Concentration Assay, Dimethylmethylene Blue Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes

doi: 10.1186/scrt466

Figure Lengend Snippet: Quantification of cytokines and glycosaminoglycan (GAG) in culture supernatants. Analysis of supernatants at days 7 and 28 of bone marrow-derived stem cells (BMSCs) (white bars), mixed cultures (BMSCs and chondrocytes in a ratio of 1:1, light grey bars), or chondrocytes (dark grey bars) monocultured (F, bars with pattern) or co- tri-cultured with osteoarthritis cartilage (FC, blank bars). Total amount of cytokines (A) interleukin-1 beta (IL-1β), (B) IL-6, and (C) IL-8 was quantified by antigen-specific enzyme-linked immunosorbent assays. (D) GAG released into the supernatant was quantified by dimethylmethylene blue (DMMB) assay. Results are presented as mean with standard deviation. * P <0.05; ** P <0.01; *** P <0.001. N = 6. Ch, chondrocytes.

Article Snippet: To determine the concentration of specific proteins in the supernatant, the human IL-1β sandwich ELISA kit (RayBiotech), IL-6 sandwich ELISA kit (R&D Systems), and IL-8 ELISA Kit (Gen-Probe, now part of Hologic, Bedford, MA, USA) were used in accordance with the instructions of the manufacturers.

Techniques: Derivative Assay, Cell Culture, Dimethylmethylene Blue Assay, Standard Deviation