sodium deoxycholate sdc  (Millipore)

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Colocalization of α−syn and tau fibrils with SDCs and flotillins. SDC transfectants, treated with fluorescently labeled α−syn or tau fibrils (at a concentration of 5 µM monomer equivalent) for 3 h at 37 °C, were permeabilized and treated with the respective APC-labeled SDC antibodies, along with either flotillin 1 (FLOT1) or 2 (FLOT2) antibodies (both Alexa Fluor 546-labeled). Nuclei of cells were stained with DAPI and colocalization was then analyzed with CLSM. (a – d) CLSM images of SDC transfectants treated with either of the fluorescent fibrils (α−syn or tau), one of the flotillin antibodies (FLOT1 or 2) and a respective APC-labeled SDC antibody. Representative images of three independent experiments are shown (e) MOC ± SEM for the overlap of SDCs with either FLOT1 or FLOT2 was calculated by analyzing 21 cellular images (7 images per sample, experiments performed in triplicate). (f) SDS-PAGE showing fluorescent α−syn or tau immunoprecipitated with either of the flotillin antibodies from extracts of stable SDC3 and SDC4 transfectants. Fluorescent α−syn and tau were detected by Uvitec’s Alliance Q9 Advanced imaging platform. Lane 1: 0.5 ug of FITC-α−syn; Lane 2: immunoprecipitate of untreated SDC3 transfectants (controls); Lane 3–4: immunoprecipitate of FITC-α−syn-treated, stable SDC3 transfectants; Lane 5: immunoprecipitate of untreated SDC4 transfectants (controls) Lane 6: immunoprecipitate of FITC-α−syn-treated, stable SDC4 transfectants; Lane 7: Molecular weight (MW) marker; Lane 8: 0.5 ug of FITC-tau; Lane 9–10: immunoprecipitates of FITC-tau-treated stable SDC3 transfectants. Lane 11: immunoprecipitate of untreated SDC3 transfectants (controls). Standard protein size markers are indicated on the left.

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Labeling, Concentration Assay, Staining, Confocal Laser Scanning Microscopy, SDS Page, Immunoprecipitation, Imaging, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Colocalization studies after treatment with the monomers. SDC transfectants (established in K562 cells) were treated with either of the fluorescently labeled α−syn, tau monomers (at a concentration of 5 µM) or Trf (25 µg/ml) for 1 h or 18 h at 37 °C. After incubation, the cells were permeabilized and treated with the respective APC-labeled SDC antibody. Nuclei of cells were stained with DAPI and cellular uptake was then analyzed with CLSM. (a – c) CLSM images of SDC transfectants 1 h after treatment with either of the fluorescently labeled monomers (α−syn, tau) or Trf. Representative images of three independent experiments are shown. Scale bar = 10 μm. (d ) Mander’s overlap coefficient (MOC) ± SEM for the overlap of SDCs with either of the fluorescently labeled monomers proteins (α−syn, tau) and Trf was calculated by analyzing 21 cellular images (7 images per sample, experiments performed in triplicate). (e – g) CLSM images of SDC transfectants 18 h after treatment with either of the fluorescently labeled monomers (α−syn, tau) or Trf. SDCs are labeled with the respective APC-labeled SDC antibody. Representative images of three independent experiments are shown. Scale bar = 10 μm. (h) MOC ± SEM for the overlap of SDCs with either of the fluorescently labeled monomers proteins (α−syn, tau) and Trf was calculated by analyzing 21 cellular images (7 images per sample, experiments performed in triplicate).

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Labeling, Concentration Assay, Incubation, Staining, Confocal Laser Scanning Microscopy

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Effect of SDC domains on α−syn and tau fibril uptake. ( a ) Flow cytometry histograms representing HS expression of WT K562 cells or SDC transfectants after NaClO 3 treatment. (b , c) Flow cytometry histograms representing intracellular fluorescence of fluorescent (FITC) fibril (α−syn or tau) treated WT K562 cells and SDC transfectants preincubated with or without NaClO 3 . (d) The effect of NaClO 3 were expressed as percent inhibition, calculated with the following formula: [(X − Y)/X] × 100, where X is the fluorescence intensity obtained on cells treated with either of the fibrils in the absence of NaClO 3 and Y is the fluorescence intensity obtained on cells treated with either of the fibrils in the presence of NaClO 3 . The bars represent mean ± SEM of four independent experiments. Statistical significance vs controls untreated with NaClO 3 was assessed by analysis of variance (ANOVA). *p

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Flow Cytometry, Cytometry, Expressing, Fluorescence, Inhibition

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Effects of SDCs on α−syn and tau fibrillation. WT K562 cells and SDC transfectants were incubated with α−syn and tau monomers at a concentration of 5 μM for up to 18 h at 37 °C. ( a,b ) Kinetics of α−syn and tau fibrillation after 1 h (a) and 18 h (b) . 1, 3, 6 and 18 h after treatment with α−syn or tau monomers, the cells were treated with ThT at a concentration of 15 μM for 10 mins and fluorescence was measured. ThT fluorescence is expressed as fold change over background ThT fluorescence. The bars represent mean ± SEM of four independent experiments. Statistical significance vs α−syn or tau-treated WT K562 cells was assessed by analysis of variance (ANOVA). *p

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Incubation, Concentration Assay, Fluorescence

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Cellular uptake of α−syn and tau fibrils into SDC transfectants. WT K562 cells and SDC transfectants were incubated with either of the FITC-labeled fibrils (α−syn or tau at a concentration of 5 µM monomer equivalent) or Trf (25 µg/ml) for 3 h at 37 °C. Cellular uptake of the fibrils and Trf was then analyzed with flow cytometry and microscopy. (a) Scanning electron microscope visualization of α−syn and tau fibrils, along with WT K562 cells and SDC transfectants treated with the fibrils at 10 min and 3 h of incubation. ( b ) Flow cytometry histograms showing intracellular fluorescence of WT K562 cells and SDC transfectants, following 3 h incubation with fluorescent α−syn or tau fibrils or Trf. (c) Detected fluorescence intensities were normalized to fibril (α−syn or tau) or Trf-treated WT K562 cells as standards. The bars represent mean ± SEM of five independent experiments. Statistical significance vs standards was assessed by analysis of variance (ANOVA). *p

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Incubation, Labeling, Concentration Assay, Flow Cytometry, Cytometry, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: Contribution of syndecans to cellular uptake and fibrillation of α-synuclein and tau

doi: 10.1038/s41598-019-53038-z

Figure Lengend Snippet: Cellular uptake of α−syn, tau into WT K562 cells and SDC transfectants following treatment with the monomers. WT K562 cells and SDC transfectants were treated with monomeric, FITC-labeled or unlabeled α−syn, tau at a concentration of 5 µM or Trf (25 µg/ml) for 1 or 18 h at 37 °C. Cellular uptake of α−syn, tau and Trf were then analyzed with flow cytometry and confocal microscopy. ( a , b ) Flow cytometry histograms showing intracellular fluorescence following 1 (a) or 18 h (b) incubation with either of the proteins (α−syn, tau or Trf). (c,d) Results of flow cytometric measurements. Detected fluorescence intensities are normalized to WT K562 cells treated with the respective fluorescent proteins (standards). The bars represent mean ± SEM of five independent experiments. Statistical significance vs protein-treated (either α−syn, tau or Trf) WT K562 cells (standards) was assessed by analysis of variance (ANOVA). **p

Article Snippet: For visualizing internalization of ThT-labeled fibrils, WT K562 cells and SDC transfectants grown on poly-D-lysine-coated glass-bottom 35-mm culture dishes were incubated with α-syn, tau at a concentration of 5 µM monomer equivalent (in DMEM/F-12 without Phenol Red) at 37 °C for 18 h, then treated with Thioflavin T (ThT, Sigma) at a concentration of 25 µM for 10 min at 37 °C and rinsed two times with ice-cold PBS.

Techniques: Labeling, Concentration Assay, Flow Cytometry, Cytometry, Confocal Microscopy, Fluorescence, Incubation

Journal: International Journal of Nanomedicine

Article Title: Use of transethosomes for enhancing the transdermal delivery of olmesartan medoxomil: in vitro, ex vivo, and in vivo evaluation

doi: 10.2147/IJN.S196771

Figure Lengend Snippet: DSC thermogram of ( A ) OLM, ( B ) PC, ( C ) SDC, ( D ) physical mixture of OLM and transethosomal components, and ( E ) TE14. Abbreviations: DSC, differential scanning calorimetry; OLM, olmesartan medoxomil; PC, phospholipid; SDC, sodium deoxycholate; TE, transethosome.

Article Snippet: L-α phosphotidylcholine from egg yolk, Span 20 (S20), Span 60 (S60), sodium deoxycholate (SDC), cellulose membrane (12,000–14,000 molecular weight cutoff), and fluorescein diacetate (FDA) were purchased from Sigma Aldrich Chemical Co. (St Louis, MO, USA).

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Transbuccal delivery of betahistine dihydrochloride from mucoadhesive tablets with a unidirectional drug flow: in vitro, ex vivo and in vivo evaluation

doi: 10.2147/DDDT.S120613

Figure Lengend Snippet: Permeation profile of BH.2HCl from different mucoadhesive buccal formulations. Abbreviations: BH.2HCl, betahistine dihydrochloride; SCH, sodium cholate hydrate; SDC, sodium deoxycholate.

Article Snippet: % acetyl content, average MN ~30,000), sodium deoxycholate (SDC), sodium cholate hydrate (SCH), acetonitrile, sodium acetate, methanol and formic acid (high-performance liquid chromatography [HPLC] grade) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: CMV infection‐induced inflammation of mucosal explants promotes sDC‐SIGN release. (A) Frozen sections were analyzed by confocal imaging 2 h after sampling (a and d), after a 7‐day subculture in DMEM (b and e), or after infection with CMV (VHL/E strain) and a 7‐day subculture in DMEM (c and f). Nuclei were counterstained with DAPI (blue), and IE/E CMV Ag (a–c) or DC‐SIGN (d–f) was stained with primary, specific antibodies, followed by incubation with Alexa 488‐conjugated goat anti‐mouse mAb. (c) The inset represents a high magnification picture of an IE/E Ag‐positive nucleus. The interface between the mucosa and the lamina propria is highlighted by white dashed lines. White arrows indicate the lumen location. (B) CMV‐infected (VHL/E strain) or noninfected mucosal explant culture supernatants were harvested at Day 0 and 7 days post‐infection (pi) and submitted to sDC‐SIGN quantification by ELISA. The results are representative of at least three independent experiments. Statistical significances are represented as P values on the graph and are identical between “Day 0‐no VHL/E” versus “Day7‐no VHL/E” and “Day 0‐no VHL/E” versus “Day 0‐with VHL/E” (ns) on the one hand and between “Day 0‐with VHL/E” versus “Day 7‐with VHL/E” and “Day 7‐no VHL/E” versus “Day 7‐with VHL/E” on the other hand. (C) Inflammation‐associated cytokines and chemokines were quantified by the CBA (BD Biosciences) in CMV‐infected (VHL/E; 600,000 pfu/cm 2 ) or LPS‐treated (100 ng/ml; 24 h) explant culture supernatants, which were harvested at different time‐points post‐treatment: 0.16 (10 min), 0.5 (30 min), 1, 14, 24, or 36 h postinfection. Human IFN‐γ, TNF‐α, CXCL‐8 (IL‐8), CXCL‐10 (IP‐10), IL‐1β, and IL‐6 were quantified simultaneously in each sample after being diluted (1/10 dilution). All of these results were representative of two independent experiments.

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Infection, Imaging, Sampling, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Crocin Bleaching Assay

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: sDC‐SIGN is functional and promotes CMV infection of MoDC. (A) Interaction between CMV gB and FLAG‐sDC‐SIGN1AT1 was assessed by dot blot under native conditions. Lysates of transiently transfected HEK cells were spotted in duplicate onto a nitrocellulose membrane according to the following order (from the top to the bottom of the membrane): mock (empty pRC/CMV vector)‐, CMV gB (respectively, with 5, 2.5, and 1.25 μg gB‐encoding plasmid, pRC/CMV‐CMVgB)‐, and FLAG‐sDC‐SIGN1AT1‐expressing cells (respectively, transfected with 5 and 0.5 μg pCDNA3.1‐FLAG‐sDCSIGN1AT1 vector). CMV gB was selectively detected with a first incubation with a FLAG‐sDC‐SIGN1AT1 solution (5 μg/ml in TBS, 0.05% Tween, 5% creamed milk), followed by a HRP‐conjugated anti‐FLAG mAb (clone M2; 1/10,000) at room temperature. Control detections were made using only the HRP‐conjugated anti‐FLAG mAb or alternatively, mAb directed against DC‐SIGN (clone 3E1, biotinylated), CMV gB (clone HCMV37), or F‐actin, respectively, revealed by a HRP‐conjugated streptavidin or goat anti‐mouse IgG. (B) A single dose of TBGFP (MOI=20) was preincubated with or without increasing amounts of FLAG‐sDC‐SIGN1AT1 (final concentrations: 400, 200, 100, 50, 25, and 12.5 ng/ml) for 30 min (4°C), prior to being added to MoDC for a further 2‐h incubation at 37°C. Cells were analyzed after 24 h by flow cytometry to determine the percentage of GFP‐positive cells (i.e., early infected cells). These experiments were performed with MoDC obtained from four distinct, healthy blood donors. (C) The same experiments were performed with MoDC (a single donor; upper panels) or FSF (lower panels) in the presence or absence of a preadsoption step on a mannan‐conjugated agarose matrix. GFP + cells (i.e., CMV‐infected cells) are indicated for each panel inside the gate containing positive cells. These results are representative of two independent experiments performed with MoDC from distinct donors. SSC, Side‐scatter.

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Functional Assay, Infection, Dot Blot, Transfection, Plasmid Preparation, Expressing, Incubation, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: sDC‐SIGN is released by DCs in the course of their differentiation. Culture supernatants from (A) MoDC or (B) CD34 + hematopoietic stem cells were tested for the presence of sDC‐SIGN by ELISA. (A and B) Asterisks indicate addition of exogenous IL‐4 to culture media. (C) Western blot analysis of cell lysates (Lanes 1–5) and (D) 10× concentrated culture supernatants (Lanes 1′–5′) obtained from MoDC generated with IL‐4/GM‐CSF (Lanes 1 and 1′), IFN‐α/GM‐CSF (Lanes 2 and 2′), IL‐13/GM‐CSF (Lanes 3 and 3′), IL‐4 (Lanes 4 and 4′), or IL‐13 (Lanes 5 and 5′). The biotin‐conjugated 3E1 mAb clone (anti‐DC‐SIGN) was used as the detection antibody.

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Generated

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: Characterization of the releasing mode and oligomerization status of MoDC‐derived sDC‐SIGN. (A) Dose‐dependent effect of Marimastat™ on mDC‐SIGN (MFI, mean fluorescence intensity: black bars, cytometric analysis) and sDC‐SIGN (ELISA) expression by 6‐day immature MoDC (open bars). (B) ELISA titration of sDC‐SIGN in membrane ghosts (m), 10× concentrated culture supernatant (sn), 10× concentrated ultracentrifuged culture supernatant (snc; 100,000 g, 2 h), and post‐ultracentrifugation pellet of the culture supernatant (p). Total protein (50 μg) was used for sDC‐SIGN titrations for all tested samples (BCA total protein quantification). (C) The oligomerization status analysis of FLAG‐sDC‐SIGN1AT1/T3 was studied by gel filtration. Fractions (500 μl‐sized), each containing 100‐μg purified FLAG‐sDC‐SIGN1AT1 (▪), FLAG‐sDC‐SIGN1AT3 (♦), and CRD alone (approximately 19 kDa; ▴; monomeric control), were separated onto a gel filtration column (GE Healthcare). Elution fractions were collected (1 ml) and analyzed further by ELISA to quantify sDC‐SIGN, except for the CRD. Indeed, CRD was devoid of the neck region recognized by the H‐200‐coating anti‐DC‐SIGN pAb. Thus, CRD was quantified in eluted fractions by BCA. The results are plotted as the sDC‐SIGN concentrations, according to the elution volume. Standard MW are indicated by black arrowheads on the top of the graph and positioned at their corresponding elution volume (thyroglobulin=670,000 kD; bovine γ‐globulin=158,000 kD; chicken OVA=44,000 kD; equine myoglobin=17,000 kD). (D) Five concentrated, FLAG‐sDC‐SIGN1AT1‐associated, peak‐surrounding fractions were analyzed by Western blot. Each fraction is characterized by its elution volume, indicated for each lane on the top of the gel. FLAG‐sDC‐SIGN1AT1 was used as a positive control (200 ng loaded in the “+” lane).

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Derivative Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Expressing, Titration, Filtration, Purification, Western Blot, Positive Control

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: sDC‐SIGN is released in higher amounts in inflammatory human body fluids. (A) sDC‐SIGN was detected in human sera from 21 healthy blood donors by Western blot analysis. Each serum (10 μl) was mixed with 2× loading buffer before migrating on a SDS‐PAGE gel (10% acrylamide) under reducing conditions. After transfer onto a nitrocellulose membrane, sDC‐SIGN was detected by immunoblotting with the mAb 3E1 (anti‐DC‐SIGN). Dashed lines indicate separation among three distinct digitalized gel pictures. (B) sDC‐SIGN was quantified in the serum of 62 healthy volunteers by ELISA. Values are indicated in ng/ml and have been displayed as a box‐and‐whisker plot, done with the GraphPad Prism software (mean serum sDC‐SIGN concentration=65.36 ng/ml; min=0 ng/ml; max=154 ng/ml; median=72 ng/ml; 25th percentile=32.41 ng/ml; 75th percentile=95.75 ng/ml). (C) BAL from patients were collected and analyzed by sDC‐SIGN ELISA. Samples were separated according to their inflammatory status [i.e., (CRP) serum

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Western Blot, SDS Page, Enzyme-linked Immunosorbent Assay, Whisker Assay, Software, Concentration Assay

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: sDC‐SIGN secretion by immature, fully differenciated MoDC is positively regulated by CXCL‐8/IL‐8 and IFN‐γ, alone or in combination. IL‐4‐starved MoDC (6 days) were cultured with increasing doses of human rCXCL‐8/IL‐8, rCXCL‐10/IP‐10, rIL‐6 (0.1, 1, 10 ng/ml), or rIFN‐γ (10, 100, 1000 UI/ml). One condition consists of a mix of CXCL‐8/IL‐8 and IFN‐γ at their respective low, intermediate, and high concentrations mentioned above. MoDC supernatants were analyzed after 48 h by ELISA to measure corresponding sDC‐SIGN quantity. All of these results were representative of at least three independent experiments. Statistical significances are represented as P values.

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: Pivotal Advance: The promotion of soluble DC‐SIGN release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells

doi: 10.1189/jlb.0710386

Figure Lengend Snippet: Cloning expression and sDC‐SIGN ELISA development. (A) Schematic representation of the full‐length DC‐SIGN‐encoding cDNA amplification by RT‐PCR. The DC‐SIGN_SM/DC‐SIGN_AS primer pair was used here to amplify all DC‐SIGN sequences denoted by the “+” sign under “Amplicon 1” (product size range from 1041 to 633 bp). Amplicons were separated on an agarose gel (1%), and DNA was labeled with ethidium bromide. (B) The legend here is identical to the previous one except for the primer pair used to amplify only TM‐containing cDNA DC‐SIGN‐encoding sequences (DC‐SIGN_SS/DC‐SIGN_AS). A ″–″ sign indicated under “Amplicon 2” means that no amplicon could be detected on the gel. M, molecular weight marker. (C) Standard curve of FLAG‐sDC‐SIGN1AT1 titration. Data was plotted as OD 450 nm (with a 570‐nm reference filter) on the y ‐axis against sDC‐SIGN concentrations (ng/ml) on the x ‐axis. OD values are representative of four distinct experiments.

Article Snippet: Both sDC‐SIGN isoforms were then purified by a two‐step affinity chromatography consisting of one passage on mannan‐conjugated agarose (elution was done with 50 μg/ml mannan in TBS), followed by a second passage on a M2 affinity resin‐loaded column at low pressure (Sigma‐Aldrich).

Techniques: Clone Assay, Expressing, Enzyme-linked Immunosorbent Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Labeling, Molecular Weight, Marker, Titration