Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Viability values (%) of 3T3 cells exposed to different concentrations of E. faecalis phage Max and E. faecium phage Zip for 24 h ( A ) Phage lysate; ( B ) Phages purified by PEG/NaCl precipitation. Viability was calculated as percentage of negative control (3T3 cells without phages). Error bars represent standard deviations from three independent experiments performed in duplicate.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Purification, Negative Control

Journal: Scientific Reports

Article Title: Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model

doi: 10.1038/s41598-019-43115-8

Figure Lengend Snippet: Efficacy of phages against bacteria colonizing 3T3 cells. ( A ) Concentration of viable bacterial cells; and ( B ) number of 3T3 cells in control (without treatment) and after 6 and 24 h of phage treatment. Error bars represent standard deviations from three independent experiments performed in duplicate. *Statistically significant (p < 0.05) conditions between control and test assays.

Article Snippet: Mouse embryonic fibroblast 3T3 cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Bacteria, Concentration Assay, Control

Journal: Frontiers in Immunology

Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

doi: 10.3389/fimmu.2018.00705

Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-mediated membrane attack complex (MAC) formation and its effect to cell signaling. (A,B) MEFs were cultured in DMEM containing 10% normal human serum (NHS) in the presence or absence of 1 µg/ml HMGB1 for 1 h at 37°C. Sublytic MAC proteins were stained using anti-C5b-9 antibody (Ab) (red) and observed by confocal microscopy. Blue: DAPI. Heat-inactivated (HI) NHS was used. Scale bar, 10 µm (A) . MEFs were incubated with 10% NHS in the presence of different concentrations of HMGB1, and then the mean relative intensity of fluorescence of 10 visual fields was calculated (B) . Error bars are mean ± SD. * P < 0.05 by Student’s paired t -test. (C) MEFs were cultured in DMEM containing 5% NHS in the presence of 1 µg/ml HMGB1 for 1 h at 37°C. To observe MAC formation, MEFs were fixed and mouse anti-C5b-9 Ab was used for immunofluorescent analysis. Cholera toxin B-FITC (CTB 0.5 µg/ml, Sigma) was utilized to observe the cell membrane lipid raft using confocal microscopy. Scale bar, 10 µm. (D) bEnd.3 cells were cultured in the presence of 5% NHS and/or 5 µg/ml HMGB1 and MAC formation was observed using Western blot analysis. (E,F) MAC formation. bEND.3 cells and LN215 cells were cocultured and incubated with DMEM containing 5% NHS in the presence or absence of 5 µg/ml HMGB1 for 16 h, and the alteration of tight junction (green line) and sublytic MAC deposition (red fluorescence, arrow) was observed by using confocal microscopy. Anti-ZO-1 Ab and anti-C5b-9 Abs were used for the study. Scale bar = 10 μm (E) . The mean relative intensity of fluorescence of six visual fields of zonal occluding (ZO) was calculated (F) . Error bars are mean ± SD. * P < 0.05 by Student’s paired t -test.

Article Snippet: Mouse embryonic fibroblasts (MEFs) (immortalized by the 3T3 protocol, purchased from HMGBiotech) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5–10% NHS 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l -glutamine under 5% CO 2 in the presence or absence of 1 μg/ml HMGB1 for 1 h at 37°C.

Techniques: Membrane, Cell Culture, Staining, Confocal Microscopy, Incubation, Fluorescence, Western Blot