cell culture medium keratinocyte growth medium 2  (PromoCell)

Journal: International Journal of Molecular Sciences

Article Title: Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells

doi: 10.3390/ijms22189920

Figure Lengend Snippet: Internalization of S. aureus and S mutans in OKF6 WT, MOCK and T2R14 KD cells. ( A ) Representative LB agar plates depicting S. aureus colonies that are internalized in OKF6 WT, MOCK and T2R14 KD cells pretreated with MPP (5 μM) and cytochalasin D (1 μg/mL). A total of 100 μL of the diluent containing bacteria was grown on LB agar plates for 24 h to form CFUs. ( B ) S. aureus CFUs from representative LB agar plates were calculated and represented in a grouped bar graph. ( C ) Immunofluorescence (IF). OKF6 cells infected with BCECF-AM (10 μM) labelled S. aureus . Cells were fixed with paraformaldehyde and labeled with TRITC-phalloidin (50 μM) and DAP1 (1:10,000 dilution). The IF image shows nucleus stained with DAPI (blue), F-actin stained with TRITC-phalloidin (red) and S. aureus stained with BCECF-AM (green). The representative images are from 3 independent experiments. ( D ) Transmission electron microscopy (TEM) images of S. aureus internalized OKF6 WT, MOCK and T2R14 KD cells. Nucleus (N), cytoplasm (C) and the red arrows in the image point to S. aureus. Scale = 2 microns. The representative images are from 1 independent experiment and captured from 5 different fields. ( E ) Representative BHI agar plates depicting S. mutans colonies that are internalized in OKF6 cells pretreated with MPP (5 μM) and cytochalasin D (1 μg/mL). A total of 100 μL of the diluent containing bacteria was grown on BHI agar plates for 24 h to form CFUs. ( F ) S. mutans CFUs from the representative BHI agar plates were calculated and represented in a grouped bar graph. The data represented in the graphs are SEM of ≥5 independent experiments. Two-way ANOVA analysis, using Tukey’s multiple comparison analysis, was performed, and the observed p -value is *** p = 0.002. ( G ) The IF image shows OKF6 cells infected with BCECF-AM (green) labeled S. mutans , and nucleus stained with DAPI (blue) and TRITC-phalloidin (red). The IF images were captured using a Nikon Ti microscope using 60X oil immersion objective. The representative images are from 3 independent experiments. ( H ) TEM images of S. mutans internalized OKF6 WT, MOCK and T2R14 KD cells. The red arrows in the image point to S. mutans. The representative images are from 1 independent experiment and captured from 5 different fields.

Article Snippet: Keratinocyte growth medium-2 (KGM-2) for OKF6 cell culture was purchased from Promo Cell (Heidelberg, Germany).

Techniques: Immunofluorescence, Infection, Labeling, Staining, Transmission Assay, Electron Microscopy, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells

doi: 10.3390/ijms22189920

Figure Lengend Snippet: Characterizing the GTP-Rac1, F/G actin levels in MOCK and T2R14 KD OKF6 cells. ( A ) The MOCK and T2R14 KD OKF6 cell lysates (500 μg) were incubated with PAK1 agarose beads (20 μg) for 1 h at 4 °C. After incubation the beads were washed, re-suspended in 2× Laemmli buffer and separated by 12% SDS-PAGE. Western blot analysis of PAK1 pull-down and total protein lysates using primary mouse monoclonal anti-Rac1 antibody (1:1000), mouse monoclonal anti β-Actin antibody (1:25,000) and secondary anti-mouse HRP antibody (1:5000 and 1:25,000) re-spectively. To visualize GTP-Rac1 that is at low levels compared to Total-Rac1, the blots were exposed for 5 min. To visualize PAK1 associated Actin and β-Actin the blots were exposed for 1 min. ( B ) Western blot analysis of F and G actin fractions isolated from MOCK and T2R14 KD cells and separated by 10% SDS-PAGE. The blots were probed using primary mouse monoclonal anti β-Actin antibody and exposed for 1 min. ( C ) The corresponding densitometry analysis of F and G actin isoforms. The data represented in the graphs are SEM of ≥3 independent experiments. One-way ANOVA analysis, using Tukey’s multiple comparison analysis, was performed, and the observed p-value is * p < 0.05. The blots were developed by using chemiluminescence detection system and imaged by using a ChemiDoc MP imaging system, Bio-Rad, Toronto, ON, Canada).

Article Snippet: Keratinocyte growth medium-2 (KGM-2) for OKF6 cell culture was purchased from Promo Cell (Heidelberg, Germany).

Techniques: Incubation, SDS Page, Western Blot, Isolation, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells

doi: 10.3390/ijms22189920

Figure Lengend Snippet: Characterizing the secretion of antimicrobial peptide hBD-2, nitrite/nitrate and IL-8/CXCL8 by ELISA. The OKF6 WT, MOCK and T2R14 KD cells were infected with S. aureus (50 MOI) and S. mutans (100 MOI) for 18 h. After infection, the supernatants were collected and filtered by using 0.25 μm nylon filter to remove cellular debris and bacteria. The supernatant was then used to determine secreted innate immune markers, such as ( A ) hBD-2, ( B ) nitrite/nitrate and ( C ) IL-8/CXCL8 as mentioned in methods. The representative grouped bar graphs were generated by using GraphPad Prism 7.0. The data represented in the graphs are SEM of ≥3 independent experiments. Two-way ANOVA analysis, using Tukey’s multiple comparison analysis, was performed, and the observed p -values are * p = 0.01, ** p = 0.004 and *** p = 0.002, ns = not significant.

Article Snippet: Keratinocyte growth medium-2 (KGM-2) for OKF6 cell culture was purchased from Promo Cell (Heidelberg, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Generated