Journal: Tissue Engineering. Part C, Methods

Article Title: Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages

doi: 10.1089/ten.tec.2021.0124

Figure Lengend Snippet: Activation of MDM in monolayer and 3D collagen hydrogels. MDM were cultured as monolayers (A) or within 3D collagen hydrogels (B) for 24 h before incubation with E. coli LPS (500 ng/10 6 MDM) for 24 h or preincubated with Dex (1 μg/mL) for 4 h before addition of E. coli LPS for 24 h, controls received vehicle only for 24 h. Gene expression was analyzed by qPCR for CD80 , CD206 , TNF-α , CXCL8 , and IL-6 , and expression calculated relative to the reference control B2M. Secretion of TNF-α (C) , CXCL8 (D) , and IL-6 (E) was measured by ELISA. Data are presented as mean ± SD with statistically significance differences determined using one-way ANOVA and compared with untreated control. * p < 0.05, ** p < 0.01, *** p < 0.005; n = 3. 3D, three-dimensional; ANOVA, analysis of variance; B2M, β2-microglobulin, Dex, dexamethasone; E. coli , Escherichia coli ; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharides; MDM, monocyte-derived macrophages; qPCR, quantitative polymerase chain reaction; SD, standard deviation. Color images are available online.

Article Snippet: Sections were incubated with mouse anti-human CD68 (0.4 μg/mL; clone KP1, Abcam ab955) followed by fluorescein isothiocyanate-conjugated goat anti-mouse F(ab) IgG (1 μg/mL, Abcam ab6669) and recombinant rabbit anti-human TNF-α (8 μg/mL, 17590-1-AP, Proteintech, UK) followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, USA) for 1 h at room temperature, with PBS washing between incubations.

Techniques: Activation Assay, Cell Culture, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Tissue Engineering. Part C, Methods

Article Title: Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages

doi: 10.1089/ten.tec.2021.0124

Figure Lengend Snippet: Immune response of MDM within an OME upon challenge with E. coli LPS. OME or MDM-OME were challenged with LPS (500 ng/10 6 MDM) for 24 h following treatment for 4 h with or without Dex (1 μg/mL). Gene expression was analyzed by qPCR for markers of macrophage polarization ( CD80 , CD206 , and CD163 ) and inflammation ( TNF-α , CXCL8 , and IL-6 ), relative to B2M for OME (A) and for MDM-OME (B) . Proinflammatory cytokine release was analyzed by ELISA for TNF-α (C) , CXCL8 (D) , and IL-6 (E) . Data are presented as mean ± SD with statistically significance differences determined using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.005; n = 3. Color images are available online.

Article Snippet: Sections were incubated with mouse anti-human CD68 (0.4 μg/mL; clone KP1, Abcam ab955) followed by fluorescein isothiocyanate-conjugated goat anti-mouse F(ab) IgG (1 μg/mL, Abcam ab6669) and recombinant rabbit anti-human TNF-α (8 μg/mL, 17590-1-AP, Proteintech, UK) followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, USA) for 1 h at room temperature, with PBS washing between incubations.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Tissue Engineering. Part C, Methods

Article Title: Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages

doi: 10.1089/ten.tec.2021.0124

Figure Lengend Snippet: TNF-α secretion by MDM in MDM-OME. MDM-OME were challenged with LPS for 24 h following pretreatment for 4 h with or without 1 μg/mL Dex+LPS; controls received no treatment. Immunofluorescence staining of tissue sections was performed for CD68 ( green ), TNF-α ( red ), counterstained for nuclei with DAPI ( blue ), and images merged (A) . Panel (B) shows a magnified image of the white square displayed in LPS-treated MDM-OME in ( A ). Scale bars = 20 μm. Color images are available online.

Article Snippet: Sections were incubated with mouse anti-human CD68 (0.4 μg/mL; clone KP1, Abcam ab955) followed by fluorescein isothiocyanate-conjugated goat anti-mouse F(ab) IgG (1 μg/mL, Abcam ab6669) and recombinant rabbit anti-human TNF-α (8 μg/mL, 17590-1-AP, Proteintech, UK) followed by Cy3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, USA) for 1 h at room temperature, with PBS washing between incubations.

Techniques: Immunofluorescence, Staining