Journal:

Article Title: Human ?-Defensins Inhibit BK Virus Infection by Aggregating Virions and Blocking Binding to Host Cells *

doi: 10.1074/jbc.M805902200

Figure Lengend Snippet: Incubation of HD5 and BKV inhibits viral binding. A, AF488-BKV (m.o.i.: 16) was incubated with H2O(black line) or 50 μg/ml of the indicated defensins (dotted line) in MEM, MEM containing 5% FBS, or PBS at 4 °C for 1 h. Vero cells in suspension were then incubated with the BKV mixture at 4 °C for 1 h and viral binding was evaluated using flow cytometry. Filled histograms represent cells in the absence of AF488-BKV. B, a 1:100 dilution of purified BKV was incubated with H2O or 50 μg/ml HD5 in the indicated media at 4 °C for 1 h. This preparation was 2-fold serially diluted across a U-bottom plate containing PBS. The HA plate was incubated with 0.5% erythrocytes in PBS at 4 °C for 16 h. A representation image of a HA plate is shown. C, Vero cells in suspension were treated with 50 μg/ml HD5 (dotted line) or an equal volume of H2O(bold line) in the indicated solutions at 4 °C for 1 h. Cells were washed and incubated with AF488-BKV (m.o.i. 16), biotintylated-SNA (lectin specific for α(2,6)-linked sialic acid), or biotintylated-MALII (lectin specific for α(2,3)-linked sialic acid) at 4 °C for 1 h. For SNA and MALII samples, cells were washed and incubated with AF488-labeled streptavidin. Fluorescence intensity was measured using flow cytometry. Filled histograms represent unstained cells.

Article Snippet: 16) of purified AF488-BKV in PBS or the lectins: 4 μg/ml biotintylated Maackia amurensis lectin II (MALII) or biotintylated Sambucus nigra lectin (SNA) (Vector Laboratories) in lectin HEPES buffer (10 m m HEPES, 150 m m NaCl, 0.1 m m MgCl 2 , 0.1 m m CaCl 2 , pH 7.2) at 4 °C for 1 h. Lectin binding was followed by washing and incubation of 2 μg/ml streptavidin-labeled Alexa Fluor 488 (Molecular Probes) at 4 °C for 1 h. Cells were washed twice with PBS and fluorescence intensity determined as above.

Techniques: Incubation, Binding Assay, Flow Cytometry, Purification, Labeling, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Sialic acid blockade in dendritic cells enhances CD8 + T cell responses by facilitating high-avidity interactions

doi: 10.1007/s00018-021-04027-x

Figure Lengend Snippet: Ac 5 3F ax Neu5Ac blocks sialic acid expression in BMDCs without affecting differentiation. A Schematic representation of the generation of BMDCs. BM cells from murine femurs and tibia were seeded into petri dishes containing culture medium and supplemented with GM-CSF and Ac 5 3F ax Neu5Ac (0–500 µM) or DMSO. On days 3 and 6, the medium was replenished with fresh medium containing GM-CSF and Ac 5 3F ax Neu5Ac (0–500 µM) or control DMSO. On day 7, the BMDCs were harvested. B Dose-dependent inhibition of sialic acid expression with Ac 5 3F ax Neu5Ac. Expression of α2-3- and α2-6-linked sialic acids or uncapped galactose/Tn antigen on BMDCs was detected using the lectins MALII, SNA-I and PNA, respectively, from left to right. The lectin binding was normalized to control untreated cells. Representative bar diagrams show mean values ± SD of three technical replicates. C Percentage of viable cells in BMDC cultures treated with different concentrations of Ac 5 3F ax Neu5Ac compared to control-treated cells. Representative bar diagrams show mean values ± SD of three technical replicates. D Expression of the DC differentiation marker CD11c on control and treated (250 µM Ac 5 3F ax Neu5Ac) BMDCs. Representative dot plots show CD11c + cells of control and treated (250 µM Ac 5 3F ax Neu5Ac) BMDCs. Mean percentages ± SD CD11c + cells of total viable cells are presented as bar diagram ( n = 2). (E) Ratio of two GM-CSF BMDC culture populations within viable and CD11c + BMDCs. Population 1 (left gate) is defined as CD11b hi MHC-II lo and population 2 (right gate) as CD11b int MHC-II lo . The gating strategy for the two populations is shown for control BMDC cultures and cultures treated with 250 µM Ac 5 3F ax Neu5Ac. Quantification of both BMDC populations is shown as a bar diagram ( n = 2) and the experiment was performed three times

Article Snippet: Carbo‐free blocking solution and biotinylated lectins MALII, SNA‐I, and PNA were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA).

Techniques: Expressing, Inhibition, Binding Assay, Marker