Journal:

Article Title: A protocol for a lung neovascularization model in rodents

doi: 10.1038/nprot.2007.536

Figure Lengend Snippet: Drawing illustrating the inflation apparatus to distend lungs initially with a 3% paraformaldehyde/0.1% gluteraldehyde solution (shown in blue). Fixative is delivered to the tracheal catheter at 23 cm H2Opressure (determined by gravity) and to the vascular catheter at 100 cm H2O (determined by positive pressure from a peristaltic pump). Note: use a standard pressure monometer to predetermine the height of the mercury column needed for the pump to generate this pressure. The catheters connect first to a luer adapter, or a luer-adapted needle, and then, via a 4-way stopcock, to the tubing delivering fixative from each reservoir. Hemostat clamps (indicated by black bars) regulate the flow of fixative through the delivery tubes from each reservoir. A gate clip (heavy black bar) positioned on the tubing from the peristaltic pump is used to obtain/regulate pressure in the vascular reservoir.

Article Snippet: 0.28 mm) Luer adapter needles (Beckton-Dickinson for rat trachea and pulmonary artery—16 gauge stub adapter, cat. no. 427561; for mouse trachea, 22G, 0.7 mm × 25.4 mm, cat. no. 305155; and for pulmonary artery, 27G, 0.4 mm × 12.7 mm, cat. no. 305109) 3-way stopcock with male luer-lock adapter (Mallinckrodt, cat. no. 91040) Sterile specimen containers, 4 oz (Fisher Scientific, cat. no. 14-375-147) Single-edged industrial blades (Ted Pella, cat. no. 121-72) Cotton gauze sponges, 2 inch × 2 inch (Fisher Scientific, cat. no. 22-362-178) Kendall Tendersorb underpads, 17 inch × 24 inch (Owens & Minor, cat. no. 3583 007105) Single-use disposable scalpels, no. 15 blade (Fisher Scientific, cat. no. 08-927-5C) Shell vials, 15 mm o.d.

Techniques:

Journal: eLife

Article Title: Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain

doi: 10.7554/eLife.79855

Figure Lengend Snippet:

Article Snippet: Mouse anti-p120-Catenin and mouse anti-Afadin primary antibodies were purchased from BD Transduction Laboratories.

Techniques: Transfection, Construct, Stable Transfection, Western Blot, Transduction, Recombinant, Mutagenesis, Sequencing, Software, Cell Culture, Lysis, Purification, Magnetic Beads, Chromatography, Protein Concentration, SDS Page, Electrophoresis

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Ago2 is present in both vesicular and nonvesicular forms in EV‐depleted serum conditions . (a) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from iodixanol cushions from the different media conditions probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the cushion DG fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4 or 5). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean+/‐SE. (b) Western blot analyses of DLD‐1 TCL, LEV, and DG fractions from iodixanol cushions from the different media conditions probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the cushion DG fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean+/‐SE. (c) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from 4 h UC to DG from EV‐depleted FBS in DMEM condition probed for Ago2, Flotillin‐1, and CD63. Middle panel : Relative intensities of the Ago2, Flotillin‐1, and CD63 bands in the DG fractions from 4 h UC to DG from EV‐depleted FBS in DMEM condition, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 4). Bottom panel : Percentages of protein bands found associated with Fractions 5–8 and 9–12. Data plotted as Mean ± SE.

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Cell culture media components affect the abundance of Ago2, hnRNP A2/B1, and EV markers in EVs . (a) Representative traces from nanoparticle tracking analysis of DKs‐8 SEVs obtained from different conditioning media. (b) Quantitation of DKs‐8 particle numbers in our SEV preparations by nanoparticle tracking analysis (n = 4). Data were shown as Mean ± SE. ns, not significant. (c) Representative TEM images of DKs‐8 SEVs obtained from the different conditioning media. Scale bar shows 200 nm. (d) Western blot assessments of DKs‐8 total cell lysate and SEVs (loading based on equal protein concentration and equal vesicle number) from the different conditioning media, probing for Ago2, hnRNP A2/B1, Flotillin, and CD63 (n = 4 or 5). Relative intensities of the Ago2, hnRNP A2/B1, Flotillin and CD63 bands of SEVs in (c), normalized to the band intensities of serum‐free DMEM. Paired t test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (e) Different concentrations of SEVs obtained from DKs‐8 cells conditioned with the different conditioning media were dotted on nitrocellulose membranes and probed with anti‐Ago2 (rat antibody) or anti‐CD63 antibodies in the presence (Tween) or absence (No Tween) of 0.1% Tween‐20. Right: Quantitation of Ago2 dots in presence or absence of Tween (n = 3).

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Cell Culture, Quantitation Assay, Western Blot, Protein Concentration

Journal: Journal of Extracellular Vesicles

Article Title: Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations

doi: 10.1002/jev2.12366

Figure Lengend Snippet: Hollow fiber bioreactors greatly affect EV populations and the distribution of RBPs on density gradients . (a) Western blot analyses of DKs‐8 TCL, LEV, and DG fractions from cushion density gradients for CDM‐HD in DMEM (serum‐free condition) and EV‐depleted FBS in DMEM probed for Ago2, hnRNP A2/B1, Flotillin‐1, and CD63. Middle panel: Relative intensities of the Ago2, hnRNP A2/B1, Flotillin‐1, and CD63 bands in the cushion density gradient fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 8 and 6, respectively). Bottom panel: Percentages of Ago2, hnRNP A2/B1 (abbreviated as A2/B1), Flotillin‐1 (abbreviated as Flot‐1), and CD63 bands found associated with Fractions 5–7, 8–10, and 11–12. Data plotted as Mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001. (b) Western blot analyses of DLD‐1 TCL, LEV, and DG fractions from cushion density gradients for the different media conditions probed for Ago2, hnRNP A2/B1, Flotillin‐1, and CD63. Middle panel: Relative intensities of the Ago2, hnRNP A2/B1, Flotillin‐1, and CD63 bands in the cushion density gradient fractions from the different media conditions, normalized to the band intensity of the highest SEV lane (either Fraction 6 or 7) (n = 5 and 4, respectively). Bottom panel: Percentages of Ago2, hnRNP A2/B1 (abbreviated as A2/B1), Flotillin‐1 (abbreviated as Flot‐1), and CD63 bands found associated with Fractions 5–7, 8–10, and 11–12. Data plotted as Mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse anti‐Flotillin‐1 (cat no. 610820) was purchased from BD BioSciences.

Techniques: Western Blot

Journal:

Article Title: Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer

doi:

Figure Lengend Snippet: EGT improves the T-cell response against HCV E2. (A) BALB/c mice receiving two doses of 50 μg of pF78E2 plasmid with (+EGT) or without EGT (−EGT) were analyzed for the induction of E2-specific cellular immunity. At 3 weeks after the boosting injection, the number of IFN-γ-secreting anti-E2 T cells was determined by ELISPOT on splenocytes from individual mice (indicated in the first column) using a pool of overlapping 20mer synthetic peptides encompassing the HCV E2 sequence (strain H) from aa 371 to 700 (E2 pool). IFN-γ-secreting CD4+ T cells were evaluated using peptide 1303, and the number of E2-specific IFN-γ-secreting CD8+ T cells was measured using peptide 1323. Two independent experiments were performed, with each one testing two different amounts of splenocytes (2.5 × 105 and 5 × 105) and two replicates for each tested amount of splenocytes. Average values were calculated, from the background level determined in the absence of peptides (typically less than 10 SFC/106 total splenocytes) was subtracted, and the result was expressed as the number of SFC/106 total splenocytes. Numbers corresponding to more than three times the background measured in control experiments without antigenic peptides were considered positive values and are indicated in boldface. (B) E2-specific CTL response in pF78E2-immunized representative mice from the +EGT (triangles) and −EGT (circles) groups. Splenocytes from immunized mice were restimulated in vitro and tested for cytotoxic activity against p815 cells pulsed with the HCV peptide 1323 (1323; closed symbols) or dimethyl sulfoxide (open symbols). Effector cell/target cell ratios are indicated in the abscissa. The percentage of specific killing is reported on the vertical axis. Each number represents the average of two independent experiments.

Article Snippet: After extensive washing (1× PBS, 0.005% Tween), biotinylated rat anti-mouse IFN-γ antibody (catalog number 18112D; PharMingen) was added and incubated overnight at 4°C.

Techniques: Plasmid Preparation, Injection, Enzyme-linked Immunospot, Sequencing, In Vitro, Activity Assay

Journal:

Article Title: Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer

doi:

Figure Lengend Snippet: EGT improves T-cell response against HIV-1 gag. BALB/c mice were injected with one (pd1) or two (pd2) doses of 5 μg of pHIV-1 gag plasmid. At 3 weeks after each injection, the number of IFN-γ-secreting anti-HIV-1 gag CD8+ T cells was determined by ELISPOT on pooled splenocytes from two immunized mice using a synthetic peptide-reproducing gag amino acid sequence between residues 197 and 205. Two different amounts of splenocytes (2.5 × 105 and 5 × 105) were tested with three replicates for each tested amount of splenocytes. Average values were calculated, the background level determined in the absence of the gag peptide (typically less than 10 SFC/106 total splenocytes) was subtracted, and the result was expressed as the number of SFC/106 total splenocytes. Black triangles represent the number of SFC/106 cells in EGT-treated mice (+EGT); open triangles indicate the IFN-γ-secreting anti-HIV-1 gag CD8+ T cells in untreated animals (−EGT). The difference between −EGT and +EGT groups are statistically significant (P < 0.01 and P < 0.0002 after one or two doses, respectively).

Article Snippet: After extensive washing (1× PBS, 0.005% Tween), biotinylated rat anti-mouse IFN-γ antibody (catalog number 18112D; PharMingen) was added and incubated overnight at 4°C.

Techniques: Injection, Plasmid Preparation, Enzyme-linked Immunospot, Sequencing