Journal: Immunity

Article Title: Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

doi: 10.1016/j.immuni.2020.01.001

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Article Snippet: ELISA plates were coated overnight at 4°C with 0.1 μg/mL of mouse anti-human IgG (human CH2 domain with no cross-reactivity to rhesus macaque IgG; clone R10Z8E9; BioRad) and then blocked for 2 h. The serum samples were assayed at 4-fold dilutions starting at a 1:100 dilution in ELISA diluent (PBS containing 1% heat-inactivated FBS and 0.2% Tween-20).

Techniques: Recombinant, Virus, Mutagenesis, Expressing, Diagnostic Assay, Staining, Plasmid Preparation, Software, Flow Cytometry, Electro Cell Manipulation, Chromatography, Microscopy

Journal: Stem Cells and Development

Article Title: Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways

doi: 10.1089/scd.2016.0199

Figure Lengend Snippet: Preparation of monocyte-derived dendritic cells (DC). (A) Plastic-adherent PBMC (>80% CD14+) was differentiated in DC medium for 5–7 days. (B) Greater than 95% of high forward versus side scatter cells in the DC differentiation culture expressed high levels of CD11c and MHCII. DC derived from adherent monocytes (see Materials and Methods section) were defined as large cells (relative high forward and side scatter profiles by flow cytometry) expressing high levels of surface CD11c and MHCII. (C) Cells expressing high surface CD11c readily phagocytosed soluble ovalbumin (OVA) antigen. Fluorescence microscopy showing cells with high surface expression of CD11c (arrow), and “OVA” arrow indicates APC-conjugated OVA that has been endocytosed by CD11chi cells. Similar results were obtained in >10 additional experiments. PBMC, peripheral blood mononuclear cell.

Article Snippet: Adherent cells were between 70% and 85% CD14 + identified by flow cytometry using the Alexa Fluor 647-conjugated (Tük4 clone, IgG2a) canine cross-reactive CD14 antibody ( ) ( www.bio-rad-antibodies.com/human-cd14-antibody-tuk4-mca1568.html ).

Techniques: Derivative Assay, Flow Cytometry, Expressing, Fluorescence, Microscopy

Journal: Blood Cancer Journal

Article Title: Role of apurinic/apyrimidinic nucleases in the regulation of homologous recombination in myeloma: mechanisms and translational significance

doi: 10.1038/s41408-018-0129-9

Figure Lengend Snippet: a (I) Antibody array : Cell Cycle Antibody Array (Hypomatrix) was sequentially treated with ARP cell lysate and HRP-conjugated anti-APEX1 antibody. Interacting partners were then identified by their location on the array. (II) Immunoprecipitation: Interaction between endogenous APEX1 and p73 was detected in the ARP cells, either control (DMSO) or those treated with 5 µM API3 for 24 h. Nuclear lysates were immunoprecipitated using anti-P73 antibody or IgG (control) and protein complexes resolved on Western blot which was probed with anti-APEX1 antibody; Input, lysate before immunoprecipitation. b (I-III): APEX1 and P73 bind to RAD51 promoter in MM cells. Protein-DNA complexes from ARP cells treated as below were immunoprecipitated using anti-P73 antibody (I and III) or anti-APEX1 antibody (II). DNA was then extracted and occupancy of RAD51 promoter evaluated by Q-PCR; Treatments : C, control (DMSO); API3, cells treated with APEX1 inhibitor 5 µM for 24 h; CS, control shRNA; APEX1-KD, APEX1-knockdown cells; (IV) Western blot showing APEX1 in CS and APEX1-KD cells. c . Inhibition of APEX1 inhibits RAD51 promoter activity in MM cells. (I) RAD51 promoter was cloned upstream of firefly luciferase gene in a plasmid, which was then introduced into ARP cells. APEX1 in these cells was inhibited by treating them with small molecule (API3, 5 µM) for 24 h, and cells evaluated for luciferase activity; (II) ARP cells transduced with lentivirus particles carrying control (CS) or APEX1-shRNA (APEX1-KD) were selected in puromycin and following confirmation of knockdown, cells were co-transfected with RAD51 promoter plasmid (described in panel I) and a plasmid carrying Gaussia luciferase as control for transfection efficiency. RAD51 promoter activity was assessed from ratio of two luciferase activities; error bars represent SDs of triplicate assays

Article Snippet: For immunoprecipitation studies, anti-APEX1 antibody (Cat. # ab194, Abcam) and anti-p73 antibody (Cat. # NBP2-24737, Novus Biologicals) were used.

Techniques: Ab Array, Immunoprecipitation, Control, Western Blot, shRNA, Knockdown, Inhibition, Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Transduction, Transfection