Journal: Biomolecules

Article Title: Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes.

doi: 10.3390/biom12071008

Figure Lengend Snippet: Figure 2. Mouse adipose stem cell (ASC) isolation and confirmation. (A). Morphology of ASC shows regular fibroblastic cell morphology. (B). Flow cytometric analysis of cultured ASC at 3rd passage. ASC was positive for CD29, CD90, and CD105 and negative for CD45, which confirms the purity of ASC. (C). Immunofluorescence staining confirmed the expression of CD29 and CD105 in ASCs. The nucleus was stained with DAPI. (D,E). Confirmation of the multipotency of ASC. (D). Adipogenic differentiation. Oblate-like cell morphology and numerous lipid droplets can be visualized without specific staining. E. Osteogenic differentiation. The cells developed a cluster-shaped structure and showed positive red staining with alizarin red staining, confirming the multipotency of ASC.

Article Snippet: The cells were then incubated with Alexa fluor 647 anti-mouse CD29 (Biolegend, Tokyo, Japan), Alexa fluor 488 anti-mouse CD105 (Biolegend), phycoerythrin anti-mouse CD90 (Novus Biologicals, Littleton, CO, USA), FITC anti-mouse CD45 (Invitrogen, Waltham, MA, USA) fluorescent conjugated antibodies or isotype control Alexa flour 647 anti-mouse IgG1(Biolegend), and FITC anti-mouse IgG1 (Biolegend) antibodies according to recommended concentration and incubated for 1 h at RT.

Techniques: Isolation, Cell Culture, Staining, Expressing

Journal: PLoS ONE

Article Title: New Agents for Targeting of IL-13RA2 Expressed in Primary Human and Canine Brain Tumors

doi: 10.1371/journal.pone.0077719

Figure Lengend Snippet: Properties of monoclonal antibodies raised against three antigenic peptides.

Article Snippet: 15 μg/milliliter of 1E10B9 mAb or isotype control mouse IgG1 (Rockland Immunochemicals) were added at designated time points.

Techniques: Bioprocessing, Enzyme-linked Immunosorbent Assay, Peptide ELISA, Western Blot, Flow Cytometry, Binding Assay

Journal:

Article Title: Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens ▿ †

doi: 10.1128/JVI.02232-07

Figure Lengend Snippet: Biological activity of immune responses elicited by the three different CCR5 peptides (cyclic WT, Ala95-95, and linear WT) in mice and chickens. (A) Flow cytometry analysis of the anti-CCR5 antibodies elicited in mice and chickens. CCR5 expression was evaluated by indirect binding of mouse and chicken antibodies in the presence or absence of 2D7 in human CD4+ cells. The relative surface expression was evaluated either as the mean fluorescence or as a percentage of CCR5 expression. The isotype controls for 2D7 (mouse IgG1-PE), rabbit anti-mouse-FITC (RAM-FITC), goat anti-chicken-FITC (GAC-FITC), NMS, and chicken and human Igs were used as negative controls. 2D7 and the isotype control coupled to PE as well as RAM and GAC coupled to FITC were used. (B) CCR5 downregulation on human and mouse CD4+ lymphocytes by 40 ng of CCR5-purified Ig from immunized mice. The CCR5 level was evaluated as the percentage of CCR5 expression by fluorescence-activated cell sorter analysis. Error bars represent standard deviations of three replicates for each data point. Assays were repeated three times with three different donors, and P values are shown. These data were obtained with serum samples drawn 1 week after the fifth immunization. (C) Inhibition of MIP-1β-induced chemotaxis of human CD4+ lymphocytes by anti-CCR5 mouse antibodies. Purified lymphocytes were incubated with Ig fractions from antisera elicited by cyclic WT, Ala95-96, or linear WT peptide. Controls included lymphocytes treated with Ig fractions from NMS and those without added antibodies (FCS). The results are expressed as the mean of the chemotaxis index. Error bars represent standard deviations for three assays for each data point. (D) Dose-response curves of the blocking of the infectivity of an HIV-1 primary isolate (HIV #36, subtype B, R5-tropic virus) by anti-CCR5 mouse antibodies. As negative controls, serum-purified total Igs from a pool of five untreated mice (NMS) were used. The data are representative of the two experiments performed. (E) Antibody-mediated CCR5 downregulation in vivo. The CCR5 phenotype of CD4+ lymphocytes was evaluated by flow cytometry of cells derived from peripheral blood of mice immunized with cyclic WT, Ala95-96, or linear WT peptide. The staining was obtained using M20 (anti-mouse polyclonal antibody). CD4+ lymphocytes from untreated mice were used as positive controls for CCR5 expression. The data are expressed as the percentage of CCR5 expression and are shown as the averages from at least three independent experiments. P values also are shown.

Article Snippet: Cells then were incubated with 2 μl of rabbit anti-mouse total Ig-FITC (Dako) or 2 μl of goat anti-chicken IgY-fluorescein isothiocyanate (FITC) (Santa Cruz) and with 5 μl of 2D7-phycoerythrin (PE) (BD Bioscience Pharmingen, San Diego, CA) for 30 min at 4°C.

Techniques: Activity Assay, Flow Cytometry, Expressing, Binding Assay, Fluorescence, Purification, Inhibition, Chemotaxis Assay, Incubation, Blocking Assay, Infection, In Vivo, Derivative Assay, Staining

Journal:

Article Title: Two Amino Acid Substitutions within the First External Loop of CCR5 Induce Human Immunodeficiency Virus-Blocking Antibodies in Mice and Chickens ▿ †

doi: 10.1128/JVI.02232-07

Figure Lengend Snippet: Characterization of chicken immune responses elicited by Ala95-96 peptide. (A) Inhibition of MIP-1β-induced chemotaxis of human CD4+ lymphocytes by anti-CCR5 chicken antibodies. Purified lymphocytes were incubated with chicken CCR5-specific IgY antibodies at concentrations ranging from 0.45 to 30 ng/ml. Lymphocytes from two healthy blood donors were used. Negative control included lymphocytes treated with IgY fractions from chicken antibodies to human Ig. Data are representative of the three assays performed. (B to H) Dose-response curves of the neutralization of the infectivity of HIV-1 strains by anti-CCR5 chicken antibodies. (B and C) Neutralization curves of SOS pseudoviruses JRFL and VSV. IgY fractions of the WT and Ala95-96 peptides were employed. As positive controls, SIM4 (anti-CD4 neutralizing monoclonal antibody) and 2D7 (anti-CCR5 neutralizing monoclonal antibody, tested at 30 μg/ml only) were used. As a negative control, IgY fractions of human Ig were tested. (D to H) HIV inhibition curves of a number of HIV-1 primary isolates. Primary isolates HIV #40 and #36 were subtype B and R5 tropic. HIV #92TH007, #92TH008, and #92BR025 were subtypes A, E, and C, respectively, and were R5 tropic, and they were provided by the AIDS Reference Research and Reagent Program. Antibodies were tested from 6 to 200 ng/ml. 2D7 (at 30 μg) and SIM4 were used as positive controls, and a chicken antibody to human Ig was used as a negative control. All data are representative of at least three of the independent assays performed.

Article Snippet: Cells then were incubated with 2 μl of rabbit anti-mouse total Ig-FITC (Dako) or 2 μl of goat anti-chicken IgY-fluorescein isothiocyanate (FITC) (Santa Cruz) and with 5 μl of 2D7-phycoerythrin (PE) (BD Bioscience Pharmingen, San Diego, CA) for 30 min at 4°C.

Techniques: Inhibition, Chemotaxis Assay, Purification, Incubation, Negative Control, Neutralization, Infection