Journal: Regenerative biomaterials

Article Title: Concentrated growth factor regulates the macrophage-mediated immune response.

doi: 10.1093/rb/rbab049

Figure Lengend Snippet: Figure 4. CGF extract modulated macrophage cytokine secretion. (A) The supernatants of CCM-treated macrophages were screened using a human cytokine anti- body array. (B) Representative cytokines are presented. (C) A GO analysis of differentially expressed cytokines enriched in BP terms after 20% CCM treatment. (D) Enriched MF categories. (E) The mRNA expression of differentially expressed cytokines (IL-1b, IL-7, RANTES and MCP-1) in CCM-treated macrophages was ana- lyzed using q-PCR. (F) The secretion of IL-1b and RANTES by CCM-treated macrophages was further confirmed using ELISAs. The quantitative data are presented as the mean 6 SD (n ¼ 3) and were analyzed using one-way analysis of variance (ANOVA) and post hoc Dunnett tests: *P< 0.05, **P< 0.01 and ***P < 0.001.

Article Snippet: The supernatants were carefully subjected to Enzyme-linked immunosorbent assays (ELISAs) for RANTES (KE00093, Proteintech Group, China) and IL-1b (KE00021, Proteintech Group, China) using a standard protocol.

Techniques: Expressing

Journal: Regenerative biomaterials

Article Title: Concentrated growth factor regulates the macrophage-mediated immune response.

doi: 10.1093/rb/rbab049

Figure Lengend Snippet: Figure 5. CGF extract regulated the macrophage-mediated immune response via the AKT pathway. (A) A western blot analysis of the proteins involved in the PI3K/AKT (p-AKT, AKT and PI3K) and JAK/STAT (p-JAK, JAK, p-STAT3 and STAT3) signaling pathways in CCM-treated THP-1 macrophages. AKT phosphorylation was increased, but no significant differences were observed in the JAK/STAT pathway (n ¼ 3). (B) The effect of an AKT inhibitor on AKT pathway inhibition was analyzed using western blotting (n ¼ 3). (C) Representative images of adherent THP-1 macrophages cultured in CCM with or without AKT inhibitor were analyzed using ImageJ software (n ¼ 5). (D) The secretion of IL-1b and RANTES by THP-1 macrophages were assayed using ELISAs (n ¼ 3). (E) Expression of the M2 sub- type marker CD163 was detected using immunofluorescence staining (n ¼ 3). The data are presented as the mean 6 SD and were analyzed using one-way analy- sis of variance (ANOVA) and post hoc tests: *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: The supernatants were carefully subjected to Enzyme-linked immunosorbent assays (ELISAs) for RANTES (KE00093, Proteintech Group, China) and IL-1b (KE00021, Proteintech Group, China) using a standard protocol.

Techniques: Western Blot, Protein-Protein interactions, Phospho-proteomics, Inhibition, Cell Culture, Software, Expressing, Marker, Staining

Journal:

Article Title: A critical role for DAP10 and DAP12 in CD8 + T cell–mediated tissue damage in large granular lymphocyte leukemia

doi: 10.1182/blood-2008-07-168245

Figure Lengend Snippet: Expression of dnDAP12(Y75A) in PBMCs from LGLL patients reduces their production of chemokines. PBMCs from LGLL patients were infected with recombinant retrovirus as described in panel B and supernatants collected at 24 hours and analyzed for (A) RANTES and (B) IL-8 production by ELISA. Viability of PBMCs was confirmed to be more than 80% at time of ELISA analyses by annexin-V–FITC labeling. ELISA results are shown from 1 representative LGLL patient; PBMCs from 2 other patients were assessed yielding similar results. The mean and SEM of triplicate wells are shown.

Article Snippet: Concentrations of RANTES and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA) as recommended by the manufacturers (Human RANTES ELISA Construction Kit [Antigenix America, Huntington Station, NY]; IL-8 BD OptEIA ELISA Kit [BD Bioscience]).

Techniques: Expressing, Infection, Recombinant, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

doi: 10.3389/fimmu.2018.00508

Figure Lengend Snippet: THP-1 cells, routinely grown in the presence of 1 mM extracellular arginine, were transfected with scrambled or SLC7A7 short interference RNA (siRNA) in the presence of 80 nM phorbol-12-myristate-13-acetate (PMA), as described in Section “ ”; after 96 h, cells were analyzed for the expression of SLC7A7 mRNA with RT-qpolymerase chain reaction (RT-qPCR) (A) and for intracellular arginine content by means of HPLC/ESI-MS-MS (B) . In the same cells, the production and release of IL1β and TNFα were monitored with RT-qPCR (C) and ELISA assay (D) , respectively. Data are mean ± SEM of four different determinations, each performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. scrambled siRNA with One sample t -test (A,C) or Student’s t -test for paired samples (B,D) .

Article Snippet: IL1β, IL8, RANTES, and TNFα in the extracellular medium were, then, quantified using Quantikine ELISA kit (R&D System), Human IL8 Picokine ELISA Kit (Vinci Biochem), Human RANTES ELISA Kit (MyBioSource), and Human TNFα ELISA Kit (Immulological Sciences), respectively.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Tandem Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

doi: 10.3389/fimmu.2018.00508

Figure Lengend Snippet: THP-1 cells, routinely grown in the presence of 1 mM extracellular arginine, were transfected for 96 h with scrambled or SLC7A7 short interference RNA (siRNA) during PMA-induced differentiation, as described in Section “ ”; when indicated, gene silencing was performed in the absence (none) or in the presence of caspase-1 specific inhibitor (z-WHED-FMK). (A–C) The expression of IL1B mRNA was determined with RT-RT-qPCR (A) , IL1β release was measured with ELISA assay (B) , and the activity of caspase-1 was determined as described in Section “ ” (C) . Data are mean ± SEM of three different determinations, each performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. scrambled siRNA; $$ p < 0.01 vs. SLC7A7 siRNA with one-way ANOVA followed by Bonferroni post hoc test. (D) The amount of NFκB-p65 in the nuclear fraction was monitored with Western Blot analysis. The experiment was repeated twice with comparative results; a representative blot is shown.

Article Snippet: IL1β, IL8, RANTES, and TNFα in the extracellular medium were, then, quantified using Quantikine ELISA kit (R&D System), Human IL8 Picokine ELISA Kit (Vinci Biochem), Human RANTES ELISA Kit (MyBioSource), and Human TNFα ELISA Kit (Immulological Sciences), respectively.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

doi: 10.3389/fimmu.2018.00508

Figure Lengend Snippet: (A,B) A549 cells were incubated in the presence of the indicated concentrations of IL1β; after 24 h, the expression of CXCL8/IL8 and CCL5/RANTES was monitored with RT-qpolymerase chain reaction (RT-qPCR). Representative experiments are shown. (C,D) A549 cells were transfected with scrambled or SLC7A7 short interference RNA (siRNA) for 96 h (see ); where indicated, IL1β (10 ng/ml) was added to the incubation medium for the last 24 h; at the end, the expression and release of the indicated chemokines was monitored at mRNA (C) and protein (D) level with RT-qPCR and ELISA, respectively. Data are mean ± SEM of three independent determinations, each performed in duplicate. * p < 0.05, ** p < 0.01 vs. scrambled siRNA; $ p < 0.05 vs. scrambled siRNA + IL1β with one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: IL1β, IL8, RANTES, and TNFα in the extracellular medium were, then, quantified using Quantikine ELISA kit (R&D System), Human IL8 Picokine ELISA Kit (Vinci Biochem), Human RANTES ELISA Kit (MyBioSource), and Human TNFα ELISA Kit (Immulological Sciences), respectively.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

doi: 10.3389/fimmu.2018.00508

Figure Lengend Snippet: A549 were incubated with fresh medium (left panels) in the presence of caspase-1 inhibitor (+ z-WHED-FMK); in parallel, another set of A549 cells (right panels) was incubated with conditioned medium (CM) of THP-1 cells (CM-THP-1) silenced for 96 h with scrambled or SLC7A7 short interference RNA (siRNA), either in the absence or in the presence of caspase-1 inhibitor. After 24 h, the expression of CXCL8/IL8 and CCL5/RANTES was monitored with RT-qpolymerase chain reaction (RT-qPCR). Data are mean ± SEM of three independent determinations, each performed in duplicate. ** p < 0.01 vs. scrambled siRNA CM-THP-1; $ p < 0.05 vs. SLC7A7 siRNA CM-THP-1 with one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: IL1β, IL8, RANTES, and TNFα in the extracellular medium were, then, quantified using Quantikine ELISA kit (R&D System), Human IL8 Picokine ELISA Kit (Vinci Biochem), Human RANTES ELISA Kit (MyBioSource), and Human TNFα ELISA Kit (Immulological Sciences), respectively.

Techniques: Incubation, Expressing, Quantitative RT-PCR