Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: International journal of molecular sciences

Article Title: Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

doi: 10.3390/ijms232012626

Figure Lengend Snippet: Figure 4. Dependence of Au/CD44/anti-CD44 complex dissociation and surface regeneration efficiency on the nature of the regeneration solution.

Article Snippet: Recombinant human CD44 biomarker (career free, His tag C-Termus), monoclonal mouse IgG clone # 2C5 (anti-CD44) and recombinant human growth hormone from E. coli (hGH) were obtained from R&D Systems (Abingdon, UK).

Techniques:

Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Increased FcγRIIIa binding of low-and hemi-fucosylated forms of JNJ-61186372. FcγRI (A), FcγRIIa (B), and FcγRIIIa (C) binding by antibodies produced in normal fucose (NF) or low fucose (LF) cell line was assessed by competitive Alpha Screen and compared to a wild type IgG1 control antibody (closed circle). JNJ-61186372 – NF (closed square), JNJ-61186372 – LF (closed up triangle), EGFR x inert arm –LF (closed down triangle), c-Met x inert arm – LF (open up triangle), EGFR (LF) x c-Met (NF) (open down triangle), EGFR (NF) x c-Met (LF) (open diamond), JNJ-61186372 – IgG2σ (open circle). Representative data from 3 replicate experiments is shown.

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Binding Assay, Produced, Control

Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Concentration of antibody (nM) to elicit 50% of maximal FcγR binding activity (IC 50 ). Data presented in mean ± SEM of 2–3 independent experiments. *Difference between JNJ-61186372 - LF and JNJ-61186372 – NF is statistically significant (p value = 0.0001).

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Concentration Assay, Binding Assay, Activity Assay, Significance Assay

Journal: mAbs

Article Title: Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

doi: 10.1080/19420862.2016.1249079

Figure Lengend Snippet: Fold change to multiple FcγR binding (IC 50 ) of different antibodies to that of JNJ-61186372 – NF.

Article Snippet: His-tagged FcγRI (R&D Systems, cat no. 1257-FC), FcγRIIa (R&D Systems, cat no. 1330-CD/CF) or FcγRIIIa (R&D Systems, cat no. 4325-FC) were captured on nickel chelate acceptor beads (PerkinElmer, cat. no. CUSM0220400EA).

Techniques: Binding Assay

Journal: Cancer immunology research

Article Title: Combined anti-VEGF and anti–CTLA-4 therapy elicits humoral immunity to galectin-1 which is associated with favorable clinical outcomes

doi: 10.1158/2326-6066.CIR-16-0385

Figure Lengend Snippet: Anti-Gal-1 antibodies isolated from an Ipi-Bev-treated patient abrogate Gal-1 binding to CD45. Anti-Gal-1 antibodies were affinity purified from plasma. Biotinylated HAS-Gal-1 (250 ng/ml) was incubated with a commercial anti-Gal-1 polyclonal antibody (Gal-1 Ab) or control antibody (10 μg/ml), enriched endogenous Gal-1 antibodies (Gal-1 Ig) or normal human IgG (1.98 μg/ml) prior to incubation with coated CD45. The binding of HAS-Gal-1 to CD45 was detected with streptavidin-HRP. Sucrose and lactose were added to the reaction at 5 mM. Data are presented as Mean ± SD of 3 experiments.

Article Snippet: Recombinant human CD45 (R&D Systems) was coated onto 96-well plates (25 ng/well) at 4°C overnight.

Techniques: Isolation, Binding Assay, Affinity Purification, Clinical Proteomics, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: CDNF promoted the proliferation of cultured NSCs. ( A ) Light-microscopic images of NSCs cultured in neurosphere on the fifth day. Scale bar = 25 μm. ( B ) The diameter of neurospheres in each group was quantified. Values represent the mean ± SEM, ** p < 0.01 according to ANOVA followed by LSD post hoc analysis.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: The effect of RADA16-I and CDNF on the differentiation of cultured NSCs. ( A ) GFAP-positive cells (green) were identified via immunohistochemical staining. DAPI (blue) was used to label the nuclei. ( B ) Tuj1-positive cells (green) were identified via immunohistochemical staining. DAPI (blue) was used to label the nuclei. ( C ) Quantitative analysis of the number of GFAP-positive cells. ( D ) Quantitative analysis of the number of Tuj1-positive cells. * p < 0.05 indicates statistical significance between treatment group and CTR group; # p < 0.05 indicates statistical significance between CDNF group and RADA16-I + CDNF group. Scale bar = 45 μm.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Cell Culture, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: RADA16-I and CDNF promoted the proliferation of NSCs and neuroblasts in the SVZ of MCAO rats. ( A ) Experimental scheme of treatment with RADA16-I and CDNF in the MCAO rats. ( B ) RADA16-I and CDNF significantly increased the number of BrdU (red)/nestin (green)-positive cells in the SVZ two weeks after MCAO. DAPI (blue) was used to label the nuclei. The insets show photomicrographs of the SVZ at higher magnification. Arrows point to the BrdU/nestin-positive cells. ( C ) Quantitative analysis of BrdU/nestin-positive cells in the SVZ. ( D ) RADA16-I and CDNF significantly increased the number of BrdU (red)/DCX (green)-positive cells in the SVZ. DAPI (blue) was used to label the nuclei. The insets show photomicrographs of the SVZ at higher magnification. Arrows point to the BrdU/DCX-positive cells. ( E ) Quantitative analysis of BrdU/DCX-positive cells in the SVZ. Every sixth section of whole SVZ in each rat was collected for staining. N = 5/group. ** p < 0.01, *** p < 0.001 according to ANOVA followed by LSD post hoc analysis. Scale bar = 45 μm. LV, lateral ventricle.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: CDNF promoted the migration of neuroblasts in the SVZ into the ischemic penumbra area of MCAO rats. ( A ) BrdU (red)/DCX (green)-positive cells were identified via immunohistochemical staining. DAPI (blue) was used to label the nuclei. The insets show photomicrographs of the ischemic penumbra at higher magnification. Arrows point to the BrdU/DCX-positive cells. Scale bar = 45 μm. ( B ) Quantitative analysis of BrdU/DCX-positive cells in the ischemic penumbra. N = 5/group. *** p < 0.001 indicates statistical significance between treatment group and CTR group; ### p < 0.001 indicates statistical significance between RADA16-I group and RADA16-I + CDNF group.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Migration, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: RADA16-I and CDNF promoted neural differentiation in MCAO rats. ( A ) BrdU (red)/GFAP (green)-positive cells were identified in the ischemic prenumbra four weeks after MCAO. DAPI (blue) was used to label the nuclei. The insets show photomicrographs of the ischemic penumbra at higher magnification. Arrows point to the BrdU/GFAP-positive cells. ( B ) Quantitative analysis of BrdU/GFAP-positive cells in the ischemic penumbra. ( C ) BrdU (red)/NeuN (green)-positive cells were identified in the ischemic prenumbra four weeks after MCAO. DAPI (blue) was used to label the nuclei. The insets show photomicrographs of the ischemic penumbra at higher magnification. Arrows point to the BrdU/NeuN-positive cells. ( D ) Quantitative analysis of BrdU/NeuN-positive cells in the ischemic penumbra. N = 5/group. ** p < 0.01, *** p < 0.001 according to ANOVA followed by LSD post hoc analysis. Scale bar = 45 μm.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: Pathways involved in the neurogenesis of CDNF and RADA16-I in MCAO rats. ( A ) Western blot analysis of p -ERK1/2 and ERK1/2. The protein levels of p -ERK1/2 were quantified relative to the level of ERK1/2. N = 4/group. ( B ) Levels of p -STAT3-Ser727 and STAT3 were determined with use of Western blotting. The protein levels of p -STAT3-Ser727 were quantified relative to the level of STAT3. N = 4/group. * p < 0.05, ** p < 0.01, *** p < 0.001 according to ANOVA followed by LSD post hoc analysis.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury

doi: 10.3390/ijms23031436

Figure Lengend Snippet: CDNF had a neuroprotective effect in brain ischemia-reperfusion injury. ( A ) TTC staining showing the infarct volume in brain sections. The representative images were placed from the anterior to the posterior portion of the brain. Scale bar = 10 mm. ( B ) The infarct volume was quantified via TTC staining. N = 5/group. ( C ) The Bederson score was assessed in rats on days 3 to 6 after MCAO. N = 10/group. ( D ) Quantitative analysis of the number of caspase-3-positive cells in the peri-infarct cerebral cortex. N = 5/group. ( E ) Cleaved caspase-3-positive cells (red) in the peri-infarct cerebral cortex. DAPI (blue) labeled the nuclei. The insets show photomicrographs of the SVZ at higher magnification. Arrows point to the caspase-3-positive cells. Scale bar = 45 μm. N = 5/group. * p < 0.05; *** p < 0.001 according to ANOVA followed by LSD post hoc analysis.

Article Snippet: Recombinant human CDNF protein (R&D Systems Inc., Minneapolis, MN, USA) was dissolved in PBS.

Techniques: Staining, Labeling

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: Lin- c-Kit+ progenitors in bone marrow express TLRs and their co-receptors.(A) TLR2, TLR4, TLR4-MD-2, or CD14 were analyzed by flow cytometry on mature peripheral cells or Lin- c-Kit+ bone marrow cells. Peritoneal lavage cells from C57BL/6 mice were stained with mAbs to F4/80 together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole spleen cells from C57BL/6 mice were stained with mAbs to B220, Mac-1, Gr-1, and CD11c together with TLR2, TLR4, TLR4-MD-2, or CD14. Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers as described in Methods and c-Kit together with TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of three independent experiments. (B) Whole bone marrow cells from C57BL/6 mice were stained with mAbs to lineage markers, Sca-1, c-Kit, and Flk-2 together with TLR2, TLR4-MD-2, or CD14. Open histograms depict staining with the isotype matched Abs. The results shown are representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Staining

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLRs and related molecules are expressed by hematopoietic stem cells and progenitor cells.(A) Lineage marker negative cells were enriched from bone marrow suspensions before staining with antibodies to hematopoietic subsets and TLR2, TLR4, TLR4-MD-2, or CD14. Open histograms depict staining with the appropriate isotype matched Abs. The results shown are representative of three independent experiments. (B) Total RNA was extracted from each progenitor subset and semi-quantitative RT-PCR was conducted to detect mRNA encoding TLR4, MD-2, TLR2, and CD14. The results are shown as values normalized to peak expression for each of the transcripts and representative of two independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Marker, Staining, Quantitative RT-PCR, Expressing

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation causes a progression of Lin+ cells from LKS+ cells and soluble CD14 augments the acquisition of Mac-1 and F4/80.(A) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 or 48 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and percentages of Lin+ or Lin- cells are indicated. (B) Sorted LKS+ cells (10,000 cells/well) from C57BL/6 mice were cultured in the presence of FL and SCF with medium alone, LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml). After 24 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers and FcγR. (C) Sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Left, open circles), Pam3CSK4 (Right, open circles) or a combination of mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles) or Pam3CSK4 (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. Data represent mean values with standard deviations from triplicate cultures (*P<0.001). The results are representative of three independent experiments. (D) Left, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with 1 μg/ml LPS (Left, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Left, filled circles). After 24, 48, or 72 hr in culture, cells were analyzed by flow cytometry for expression of lineage markers. The graph depicts cell yields and the results are representative of two independent experiments. Right, sorted LKS+ cells from C57BL/6 mice were cultured in the presence of FL and SCF with a range of concentrations of LPS (Right, open circles) or a combination of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS (Right, filled circles). After 72 hr in culture, cells were analyzed by flow cytometry for expression of Mac-1 and F4/80. The graph depicts cell yields and the data are representative of two independent experiments. (E) Sorted Flk-2- or Flk-2+ LKS+ cells (10,000 cells/well) were cultured with medium alone, LPS (10 μg/ml) or Pam3CSK (1 μg/ml). Flk-2- LKS+ cells were cultured with SCF. Flk-2 LKS+ cells were cultured with SCF and FL. After 72 h in culture, cells were analyzed by flow cytometry for expression of lineage markers (left panels). Percentages given in quadrants indicate the frequencies of Lin+ or Lin- cells and the bar graphs on the right depict cell yields. The data represent mean values with standard deviations from triplicate cultures and the results are representative of three independent experiments. (*P<0.03: Flk-2- LKS+, *P<0.002: Flk-2+ LKS+)

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Flow Cytometry, Expressing, Recombinant

Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis.(A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88-/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam3CSK4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19+ cells or Mac-1+ cells (Right) and the data represent mean values with standard deviations from triplicate cultures (*P<0.02). The results are representative of those obtained in five independent experiments. (B) Subsets of the recovered cells described in panel (A) were sorted and used to prepare Giemsa-May-Grünwal stained slides. (C) Cultured cells from C57BL/6 mice were also analyzed by flow cytometry for expression of Gr-1 and CD11c. (D) Sorted CLPs from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with a range of concentrations of LPS or combinations of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graph depicts cell yields for CD19+ cells or Mac-1+ cells (Right). Data represent mean values with standard deviations from triplicate cultures (*P<0.003). The results are representative of three independent experiments.

Article Snippet: Reagents Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Expressing, Staining, Cell Culture, Recombinant

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Frequency, based on NGS analysis, of discovered clones in the different strategies including the input phage pool. Median frequency is shown with red bars. (A) Antibodies, previously discovered through direct screening with corresponding specificities, CD23 ( n = 107), CD72 ( n = 132), and CD200, CD21, CD32, or HLA-DR ( n = 34). (B) Antibodies, discovered through fishing, binding FCRL5, ROR1, or CD22 ( n = 17). For statistical analysis, Friedman’s test with Dunn’s multiple comparison was done using GraphPad Prism.

Article Snippet: The extracellular domain of CD72 (R&D Systems, 5405-CD), CD21 (Sino Biological, 10811-H08H), and CD32 (in-house produced) were coupled to Tosylactivated M280 beads (Thermo Fisher Scientific) (200 pmol protein to 60 μl beads), according to manufacturer’s instructions and coated to an immunotube (Thermo Fisher Scientific, 470319), 70 pmol/antigen, overnight.

Techniques: Clone Assay, Binding Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: NGS analysis of identified antibodies binding to (A) CD72 ( n = 301) or (B) CD21 ( n = 27) showing the frequency in control (track C) versus antibody blocking (track B1). Yellow-marked clones were analyzed for binding, as scFv displayed on phage, to coated (C) CD72 or (D) CD21 protein in ELISA with or without prior blocking with the pool of IgGs used for blocking in tracks B1 and B2. As control, two additional clones per target, corresponding to sequences showing a decreased frequency in tracks B1 and B2 in the NGS analysis, were included. Binding was detected using an HRP-labeled anti-M13 antibody and a luminescent substrate.

Article Snippet: The extracellular domain of CD72 (R&D Systems, 5405-CD), CD21 (Sino Biological, 10811-H08H), and CD32 (in-house produced) were coupled to Tosylactivated M280 beads (Thermo Fisher Scientific) (200 pmol protein to 60 μl beads), according to manufacturer’s instructions and coated to an immunotube (Thermo Fisher Scientific, 470319), 70 pmol/antigen, overnight.

Techniques: Binding Assay, Control, Blocking Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Labeling

Journal: American journal of hematology

Article Title: A specific and sensitive assay for blood levels of glycated CD59: a novel biomarker for diabetes.

doi: 10.1002/ajh.23478

Figure Lengend Snippet: Figure 1. Specificity of the anti-glycated hCD59 rabbit monoclonal antibody (MABGlyCD59). A. GCD59 (G) and NGCD59 (NG) affinity purified from a urine pool of indi- viduals with poorly controlled diabetes, separated by SDS-PAGE and immuno-blotted with goat polyclonal anti-hCD59 antibody before (non-reduced) and after (reduced) treatment with Na-borohydride (left panels in the reduced and non-reduced frames depicting red fluorescent bands). The GCD59 band is also recognized by the MAB- GlyCD59 antibody but only after Na-borohydride reduction (green fluorescent band in the G lane of the Reduced frame). The full-length blotted membranes used to cre- ate this figure are shown in Supporting Information Fig. S1. B. Affinity purified hCD59 from a group of individuals with and without diabetes separated by SDS-PAGE and immunoblotted first with goat polyclonal anti-CD59 antibody (Goat Poly Anti-CD59; red fluorescence) and then with MABGlyCD59 antibody (green fluorescence). The bar graph at the bottom of Fig. 1B depicts the ratio of green (GCD59) over red (total CD59) fluorescence (quantified using an infrared Odyssey scanner) for each individual sample. The inset shows the mean 6 SEM of the ratios for the individuals with (D) and without diabetes (N).

Article Snippet: Hybridoma supernatants were initially selected by ELISA screening against the antigen peptide, and final clones were selected by titration against recombinant human CD59 (R&D Systems), following established protocols [18].

Techniques: Affinity Purification, SDS Page, Fluorescence

Journal: American journal of hematology

Article Title: A specific and sensitive assay for blood levels of glycated CD59: a novel biomarker for diabetes.

doi: 10.1002/ajh.23478

Figure Lengend Snippet: Figure 2. Specificity of the ELISA for GCD59. Sandwich ELISA of affinity purified GCD59 before and after immunoprecipitation (IP) with increasing concentrations of goat polyclonal anti-CD59 antibody. The insets show the Western Blot (WB) analysis for total (red fluorescence) and GCD59 (green fluorescence) of the sam- ple before (inset at 0 antibody concentration) and after immunopreciptiation (IP) with 12 mg/ml antibody (S 5 WB of the supernatant after IP; E 5 WB of eluate from agarose beads carrying the antibody-CD59 complex).

Article Snippet: Hybridoma supernatants were initially selected by ELISA screening against the antigen peptide, and final clones were selected by titration against recombinant human CD59 (R&D Systems), following established protocols [18].

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Affinity Purification, Immunoprecipitation, Western Blot, Fluorescence, Concentration Assay