Journal: Virology

Article Title: Effects of Tumor Necrosis Factor on Viral Replication and Pulmonary Inflammation during Acute Mouse Adenovirus Type 1 Respiratory Infection

doi: 10.1016/j.virol.2020.05.004

Figure Lengend Snippet: (A) Mouse 3T12 fibroblasts were infected (MOI=1) with MAV 1.pIXeGFP, a recombinant GFP-expressing MAV-1, in the presence of the indicated concentrations of TNF or vehicle control (PBS). Accumulating fluorescence expressed as relative fluorescence units (RFU) was used as an indicator of viral replication (mean ± S.E.M., n=6 per condition combined from two identical experiments). The bottom horizontal dashed line represents background fluorescence in wells with uninfected cells. Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests; *P<0.05 and ***P<0.001 compared to vehicle at a given time point. (B) Mouse 3T12 fibroblasts were infected with MAV-1 (MOI=1) in the presence of the indicated concentrations of TNF or vehicle control. RT-qPCR was used to quantify MAV-1 TPL mRNA levels at 72 hpi. Data are shown in arbitrary units standardized to GAPDH (mean ± S.E.M., n=6 per condition combined from two identical experiments). Statistical comparisons were made using one-way ANOVA followed by Kruskal-Wallis tests; ***P<0.001 compared to vehicle. (C) Mouse 3T12 fibroblasts were infected with MAV-1 (MOI=1) or mock-infected with conditioned media in the presence of the indicated concentrations of TNF or vehicle control. Cytotoxicity was assessed at 48 hpi using a luminescent cell viability assay. Data are expressed as the percentage of signal in wells with uninfected, untreated cells (mean ± S.E.M., n=9 per condition combined from three identical experiments). Statistical comparisons were made using two-way ANOVA followed by Bonferroni’s multiple comparison tests; *P<0.05 and **P<0.01 comparing mock to infected at a given concentration. There were no statistically significant differences between concentrations within a condition (mock or infected).

Article Snippet: In vitro infection Mouse 3T12 fibroblasts (ATCC CCL-164) were grown in Dulbecco’s Modified Eagle Medium (Gibco) containing 5% heat-inactivated calf serum and 1% penicillin and streptomycin (Gibco).

Techniques: Infection, Recombinant, Expressing, Control, Fluorescence, Comparison, Quantitative RT-PCR, Cell Viability Assay, Concentration Assay

Journal: Immunological Investigations

Article Title: Functional Characterization of N297A, A Murine Surrogate for low-Fc Binding Anti-Human CD3 Antibodies

doi: 10.1080/08820130802608238

Figure Lengend Snippet: N297A did not bind to Fc gamma receptors. (A) RAW264.7, a mouse macrophage cell line which highly expresses Fcγ receptors, and (B) 3T12, a non-Fcγ receptor-expressing mouse cell line, were used to analyze binding of antibodies. Serial dilution of mouse antibodies IgG2a (□), IgG2b (▴), IgG1 (▾), WT (•) and N297A (○) were incubated with the cells for 20 minutes on ice. After washing, samples were incubated with secondary APC-conjugated goat anti-mouse IgG, F(ab′) 2 fragment specific antibody (2.5 μg/ml) for detection by flow cytometry.

Article Snippet: Both RAW264.7 and 3T12 cell lines were purchased from ATCC (Bethesda, MD).

Techniques: Expressing, Binding Assay, Serial Dilution, Incubation, Flow Cytometry