Journal: Investigative Ophthalmology & Visual Science

Article Title: Therapeutic Effects of a Novel Agonist of Peroxisome Proliferator-Activated Receptor Alpha for the Treatment of Diabetic Retinopathy

doi: 10.1167/iovs.16-21402

Figure Lengend Snippet: Effects of Y-0452 on retinal vascular inflammation and leakage in diabetic rats. Rats with 1 month of STZ-induced diabetes received daily intraperitoneal injections of Y-0452 (10 mg/kg/d) or the same volume of the vehicle (Veh) as a control for 3 weeks. Retinal vascular endothelial cells and adherent leukocytes were stained with FITC–concanavalin-A and visualized under a fluorescence microscope. (A–C) Representative images of retinal adherent leukocytes in nondiabetic and STZ-induced diabetic rats ([A] nondiabetic, [B] STZ+Veh, [C] STZ+Y-0452). (D) Quantification of retinal adherent leukocytes was performed from ×40 magnification images. Adherent leukocytes were counted in six random fields per retina (white arrows indicate adherent leukocytes). (E) Retinal vascular leakage was measured using Evans blue as a tracer and normalized by total retinal protein concentrations (n = 6; *P < 0.05, **P < 0.01, 2-way ANOVA).

Article Snippet: FITC-conjugated concanavalin A (conA) (200 μg/mL in PBS, pH 7.4, 5 mg/kg body weight; Vector Laboratories) was then perfused to label adherent leukocytes and vascular endothelial cells.

Techniques: Staining, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: G-CSF inhibits LFA-1-mediated CD4 + T cell functions by inhibiting Lck and ZAP-70

doi: 10.18632/oncotarget.18194

Figure Lengend Snippet: Purified CD4 + T cells were added to 24-well plates and incubated in RPMI alone or in RPMI containing G-CSF or G-CSF plus ConA for 24 h. (A) The expression of G-CSFR on CD4 + T cells, evaluated by CD114 staining. (B) The percentage of G-CSFR is summarized in the graph (n=5 per group). After incubation 24h, G-CSF stimulated or left un-stimulated CD4 + T cells were transferred to 96-well plates coated with ICAM-1 and anti-CD3. (C) Cell morphology was examined by fluorescence microscopy at 60 minutes. Arrows indicate polarized T cells with a clear uropod. (D) Cells prepared as in C were quantified. (E) Unbound cells were washed off, and the adherent cells were quantified using a CCK-8 assay kit. The data are represented as the mean ± SD (n=15 per group). * P < 0.05; *** P < 0.001.

Article Snippet: Concanavalin A (ConA) was purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA).

Techniques: Purification, Incubation, Expressing, Staining, Fluorescence, Microscopy, CCK-8 Assay

Journal: Oncotarget

Article Title: G-CSF inhibits LFA-1-mediated CD4 + T cell functions by inhibiting Lck and ZAP-70

doi: 10.18632/oncotarget.18194

Figure Lengend Snippet: Purified CD4 + T cells were added to 24-well plates and incubated in RPMI alone or in RPMI containing G-CSF or G-CSF plus ConA for 24 h. After the incubation, the cells were transferred to anti-CD3- and ICAM-1-coated 6-well plates and incubated for 1 h at 37°C. (A and B) Lysates from the three groups of CD4 + T cells were analyzed by western blot to detect the expression of Lck, ZAP-70 and phosphorylated Lck and ZAP-70, which were normalized to actin. (C-F) Densitometry quantification was performed and presented. All data are represented as the mean ± SD (n=4 per group). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Concanavalin A (ConA) was purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA).

Techniques: Purification, Incubation, Western Blot, Expressing

Journal: bioRxiv

Article Title: A library of structurally homogeneous human N -glycans synthesized from microbial oligosaccharide precursors

doi: 10.1101/118216

Figure Lengend Snippet: (a) Structures of reference compounds that were derivatized with AEAB and immobilized onto NHS-activated glass slides, alongside glycans 2-21 . (b) Probing of immobilized glycans with biotinylated ConA (10 μg/ml). The amount of bound ConA was determined by streptavidin-Cy5 (5 μg/ml) fluorescence. Background subtracted mean fluorescence values are shown. Error bars represent the standard deviation of the mean. Representative structures for glycans 3 , 12 , 15 and 19 are shown. All glycans were printed at 100 μM in PBS in replicates of four. PBS and streptavidin-Cy5 spots served as controls. Biotin-LC-hydrazide spots printed on each subarray serve as an alignment feature to localize each subarray on the slide.

Article Snippet: Biotinylated ConA (Vector Labs) lectin binding to glycans on the array was detected by a secondary incubation with streptavidin-cyanine 5 at 5 μg/ml (SA-Cy5).

Techniques: Fluorescence, Standard Deviation