Journal: International journal of molecular sciences

Article Title: Roles of 4'- O -Methylalpinum Isoflavone on Activation of Microglia Induced by Oxysterols.

doi: 10.3390/ijms252312743

Figure Lengend Snippet: Figure 1. Effects of oxysterols on the expression of IL-6 in microglia. HMC3 microglial cells (1 × 106 cells/well) were cultured with 1 µg/mL of cholesterol and the oxysterols in a 60 mm culture dish for 48 h. (A) The transcript of IL-6 was analyzed with RT-PCR and real-time PCR. The y-axis values represent fold increases in IL-6 mRNA levels normalized to GAPDH levels relative to that of the untreated microglia (control). *** p < 0.001 vs. control. (B) After separating the supernatant of the culture medium, the levels of IL-6 protein in the media were measured by ELISA. * p < 0.05 vs. control; *** p < 0.001 vs. control. Data are expressed as the mean ± SD (n = 3 replicates for each group). This experiment was independently performed three times for each condition.

Article Snippet: The concentration of IL-6 secreted into the culture media was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Human IL-6 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Roles of 4'- O -Methylalpinum Isoflavone on Activation of Microglia Induced by Oxysterols.

doi: 10.3390/ijms252312743

Figure Lengend Snippet: Figure 2. Effects of mAI on the expression of IL-6 in microglia. HMC3 cells were stimulated with 1 µg/mL of 25OHChol and 27OHChol in the absence or presence of 1 µg/mL mAI for 48 h. (A) Using total RNA isolated from cells, IL-6 transcripts were detected by RT-PCR and real-time PCR. The y-axis values represent increases in IL-6 mRNA levels normalized to GAPDH levels relative to that of the untreated microglia (control). *** p < 0.001 vs. control.; ### p < 0.001 vs. 25OHChol plus mAI; +++ p < 0.001 vs. 27OHChol plus mAI. (B) The levels of IL-6 protein secreted from the cells in the cultured media were measured by ELISA. The data are expressed as the mean ± SD (n = 3 replicates for each group). *** p < 0.001 vs. control.; ### p < 0.001 vs. 25OHChol plus mAI; +++ p < 0.001 vs. 27OHChol plus mAI. This experiment was independently performed three times for each condition.

Article Snippet: The concentration of IL-6 secreted into the culture media was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Human IL-6 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: Roles of 4'- O -Methylalpinum Isoflavone on Activation of Microglia Induced by Oxysterols.

doi: 10.3390/ijms252312743

Figure Lengend Snippet: Figure 4. Effects of mAI on the expression of IL-1β induced by the oxysterols in microglia. The cells (1 × 106 cells) were cultured in a 60 mm culture dish with 1 µg/mL of cholesterol and the oxysterols for 48 h. (A) Total RNA was isolated from the cells, and the transcript of IL-1β was analyzed with RT- PCR and real-time PCR. The y-axis values represent fold increases in IL-1β mRNA levels normalized to GAPDH levels relative to that of the untreated microglia (control). *** p < 0.001 vs. control; ### p < 0.001 vs. 25OHChol or 27OHChol. (B) After separating the supernatant of the stimulated cells, the levels of IL-1β protein in the media were measured by ELISA. *** p < 0.001 vs. control; ### p < 0.001 vs. 25OHChol or 27OHChol. Data are expressed as the mean ± SD (n = 3 replicates for each group).

Article Snippet: The concentration of IL-6 secreted into the culture media was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Human IL-6 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Inflammatory responses in H69 cells induced by LPS-derived EVs. ( a ) Analysis of inflammatory responses. H69 cells were incubated with PBS- or LPS- derived EVs for 48 hours. Total RNAs were harvested and two-step RT-PCR was performed (n = 5). For ELISA (n = 10) and cell proliferation assay (n = 10), H69 cells were cultured in 10 cm dishes or 96-well plates, respectively and incubated with EVs for 48 hours. Analyses were performed using commercial kits according to manufacturers’ instructions. ( b ) EV disruption abolished the effects of EVs. EVs were boiled at 95 °C for 15 min and incubated with H69 cells. After 48 hours, RT-PCR analysis for IL-1β and IL-6 (n = 3) and proliferation assay (n = 10) were performed. ( c ) H69-derived EVs did not induce inflammatory responses in primary hepatocytes. Human primary hepatocytes were incubated with PBS- or LPS-derived EVs isolated from H69 cells for 48 hours. RT-PCR was performed to analyze cytokine production (n = 10).

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Derivative Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Cell Culture, Isolation

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Inflammatory cell-cell communication between large cholangiocytes. ( a ) Small and large cholangiocytes were incubated with PBS- or LPS-derived EVs isolated from small cholangiocytes for 48 hours. RT-PCR for proinflammatory cytokine expression (n = 6) and cell proliferation assay (n = 10) were performed. ( b ) Small and large cholangiocytes were incubated with EVs isolated from large cholangiocytes for 48 hours. RT-PCR (n = 6) and cell proliferation assay (n = 10) were performed.

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Incubation, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Proliferation Assay

Journal: Scientific Reports

Article Title: The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

doi: 10.1038/s41598-017-10694-3

Figure Lengend Snippet: Secretion of EVs and responses against LPS in large cholangiocytes with SCT or SR knockdown. ( a ) The morphological analysis by transmission electron microscope for EVs isolated from control large cholangiocytes (left), with SCT knockdown (middle), and with SR knockdown (right). Scale bar: 100 nm. ( b ) Nanoparticle tracking analysis for isolated EVs. An example for PBS- (solid lines) and LPS-derived (dashed lines) EVs isolated from control (blue lines), SCT knockdown (red lines), and SR knockdown (green lines) cholangiocytes is shown (top). Total EV concentrations are shown (bottom). * P < 0.05, ** P < 0.001 vs. control cholangiocytes with same stimulation (n = 5). ( c ) Knockdown efficiencies for SCT and SR. Control, SCT or SR knockdown cholangiocytes were incubated with 50 ng/mL LPS for 72 hours in 6-well plates. Culture media were harvested for ELISA for SCT, and total RNAs were harvested from cells for RT-PCR for SR. * P < 0.05, ** P < 0.001 vs. control cholangiocytes (n = 3). ( d ) For RT-PCR for proinflammatory cytokine expression, cholangiocytes were incubated with 1× PBS or 200 ng/mL LPS for 3 hours. Total RNAs were harvested and two-step RT-PCR was performed (n = 6). For cell proliferation assay, cholangiocytes were incubated with 1× PBS or 200 ng/mL LPS for 48 hours (n = 10).

Article Snippet: IL-6 in culture media was detected using Human IL-6 ELISA Kit II (BD Biosciences, San Jose, CA).

Techniques: Transmission Assay, Microscopy, Isolation, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Proliferation Assay

Journal: iScience

Article Title: Salivary exosomes exacerbate colitis by bridging the oral cavity and intestine

doi: 10.1016/j.isci.2024.111061

Figure Lengend Snippet: Effect of salivary exosomes on mucosal inflammation and intestinal barrier integrity (A) Co-culture model of salivary exosomes and Caco-2 cells in vitro . (B) The ZO-1 expression in Caco-2 cells was evaluated by western blot. (C–E) Protein levels of cytokines IL-6, TNF-α, and IL-1β were assessed in the cell supernatant of Caco-2 cells using ELISA. IL-6: UC/A vs. LPS: ∗, UC/R vs. LPS: ns, CD/A vs. LPS: ∗∗∗∗, CD/R vs. LPS: ns, HC vs. LPS: ns; TNF-α: UC/A vs. LPS: ∗∗, UC/R vs. LPS: ∗, CD/A vs. LPS: ∗∗∗∗, CD/R vs. LPS: ∗, HC vs. LPS: ns; IL-1β: UC/A vs. LPS: ∗, UC/R vs. LPS: ns, CD/A vs. LPS: ∗∗, CD/R vs. LPS: ns, HC vs. LPS: ns. (F and G) Representative western blotting of zo-1 DSS-induced mouse guts, and quantification of ZO-1 expression normalized to β-actin. UC/A vs. PBS: ∗∗∗, UC/R vs. PBS: ns, CD/A vs. PBS: ∗, CD/R vs. PBS: ns, HC vs. PBS: ns. (H and I) Western blot analysis of caspase-3 in the intestines of DSS mice, with quantification of caspase-3 protein expression levels standardized to β-actin. UC/A vs. PBS: ∗∗∗, UC/R vs. PBS: ns, CD/A vs. PBS: ∗, CD/R vs. PBS: ∗, HC vs. PBS: ns. Data were presented as means ± SEM. ns: p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human IL-6 ELISA Kit , MultiSciences , Cat# 70-EK106/2-24.

Techniques: Co-Culture Assay, In Vitro, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Salivary exosomes exacerbate colitis by bridging the oral cavity and intestine

doi: 10.1016/j.isci.2024.111061

Figure Lengend Snippet: Impact of saliva-derived exosomes on THP-1 cells (A) Co-culture model of salivary exosomes alongside THP-1 cells in vitro . (B–D) Protein quantities of the cytokines IL-1β, IL-6, and TNF-α in the cell supernatant of THP-1 cells assessed via ELISA. (E and F) mRNA levels of M1 macrophage makers CD80 and CD86 were measured using real-time qPCR (normalized to GAPDH). Data were presented as means ± SEM. ns: p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human IL-6 ELISA Kit , MultiSciences , Cat# 70-EK106/2-24.

Techniques: Derivative Assay, Co-Culture Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Salivary exosomes exacerbate colitis by bridging the oral cavity and intestine

doi: 10.1016/j.isci.2024.111061

Figure Lengend Snippet:

Article Snippet: Human IL-6 ELISA Kit , MultiSciences , Cat# 70-EK106/2-24.

Techniques: Recombinant, Saline, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Gel Extraction, Sequencing, Real-time Polymerase Chain Reaction, Software

Journal: Aging (Albany NY)

Article Title: Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer

doi: 10.18632/aging.202289

Figure Lengend Snippet: Pharmacological blockade of FASN activity modifies autocrine prolactin secretion in a PR-dependent manner. ( A ). Top. Autocrine prolactin secretion levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of graded concentrations of C75 in 0.5% CCS for 48 h were determined by a commercially available EASIA kit. Data are means ( columns ) ± S.D. ( bars ) from three independent experiments performed in duplicate. Secreted amounts of prolactin in C75-treated cells were compared with those in vehicle-treated control cells (* P < 0.05; ** P < 0.005). Bottom. Cell lysates strictly obtained from the same experimental replicates employed in A were subjected to immunoblotting for PRLR protein expression. β-actin was used to control for protein loading and transfer. Figure shows a representative immunoblot analysis. Similar results were obtained in 3 independent experiments. ( B ). Autocrine IL-6 levels in the extracellular milieu of estradiol-depleted cells cultured in the absence or presence of 10 μg/mL C75 in 0.5% CCS for 48 h were determined by a commercially available ELISA kit. Secreted amounts of IL-6 in C75-treated cells were compared with those in vehicle-treated control cells (** P < 0.005).

Article Snippet: The amount of IL-6 conditioned media was determined with the Human IL-6 Quantikine ELISA Kit (catalog #D6050; R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Cell Culture, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Reports

Article Title: Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells

doi: 10.1016/j.stemcr.2018.07.009

Figure Lengend Snippet: Expression of Cytokines by hESC-RPE Cells (A) Real-time PCR analysis of the expression of cytokines by RPE cells cultured for 3 days in the presence or absence of IFN-γ. Relative quantity is the relative expression level of each gene in comparison with its expression level in unstimulated RPE cells that was set at 1. (B) ELISA analysis of the secretion of cytokines, IL-6, IL-18, and IL-15 by RPE cells cultured for 3 days in the presence or absence of IFN-γ. (C) RT-PCR analysis of the expression of IL-12A, IL-6, IL-15, and IL-18 by RPE cells cultured for 3 days with or without IFN-γ. Data are presented as means ± SEM of at least 3 independent experiments. ∗∗∗ p ≤ 0.001.

Article Snippet: The amount of cytokines in the samples collected from RPE cells and from PBMCs cultured w/or w/o RPE cells was quantified using commercial human ELISA systems (human IL-15, R&D Systems; human IL-6, IL-10, IL-2, and IFN-γ, Ready-Set-Go, eBioscience; IL-18, Medical and Biological Laboratories, Nagoya, Aichi, Japan).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Reports

Article Title: Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells

doi: 10.1016/j.stemcr.2018.07.009

Figure Lengend Snippet: Decrease in Secretion of IFN-γ and Increase in Secretion of IL-10 and IL-2 by PBMCs following Co-culture with hESC-RPE Cells ELISA analysis of the secretion of IFN-γ (A), IL-10 (B), and IL-2 (C) by activated PBMCs that were co-cultured for 1–4 days with RPE cells. (C) At day 4 no secretion of IL-2 by control PBMCs was detected (n = 2). Data are presented as means ± SEM of at least 3 independent experiments. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Article Snippet: The amount of cytokines in the samples collected from RPE cells and from PBMCs cultured w/or w/o RPE cells was quantified using commercial human ELISA systems (human IL-15, R&D Systems; human IL-6, IL-10, IL-2, and IFN-γ, Ready-Set-Go, eBioscience; IL-18, Medical and Biological Laboratories, Nagoya, Aichi, Japan).

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: Stem Cell Reports

Article Title: Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells

doi: 10.1016/j.stemcr.2018.07.009

Figure Lengend Snippet: Transplanted hESC-RPE Cells Provide Functional Retinal Rescue in the Absence of Systemic Immunosuppression (A) Color fundus photographs show subretinal grafts of pigmented cells (marked by arrows) in RCS rats in the presence (upper image) or absence (lower image) of systemic cyclosporine immunosuppression 5–6 weeks post-transplantation. (B) Mean b-wave amplitudes in response to white flashes of increasing intensity. The mean b-wave amplitudes were significantly higher both in cyclosporine-treated (red line) and non-treated (green line) animals compared with non-transplanted fellow eyes (red and green dashed lines, respectively). (C) Representative H&E-stained retinal sections show relative preservation of photoreceptors layer (ONL) in proximity to subretinal RPE grafts in cyclosporine-treated and non-treated animals, in comparison with a thinner ONL in a non-transplanted eye. (D) Anti-GFP immunostaining show presence of transplanted cells in the subretinal space of RCS rats either treated or untreated with systemic immunosuppression. (E) ELISA analysis of the peripheral blood IL-10 levels in RCS rats 4–5 weeks post-transplantation. Levels of anti-inflammatory interleukin IL-10 were statistically higher in non-cyclosporine group compared with the cyclosporine-treated group and non-transplanted control. ∗ p ≤ 0.05. Data are presented as means ± SEM of at least 3 independent experiments.

Article Snippet: The amount of cytokines in the samples collected from RPE cells and from PBMCs cultured w/or w/o RPE cells was quantified using commercial human ELISA systems (human IL-15, R&D Systems; human IL-6, IL-10, IL-2, and IFN-γ, Ready-Set-Go, eBioscience; IL-18, Medical and Biological Laboratories, Nagoya, Aichi, Japan).

Techniques: Functional Assay, Transplantation Assay, Staining, Preserving, Comparison, Immunostaining, Enzyme-linked Immunosorbent Assay, Control