Journal:

Article Title: IL-15-induced conversion of monocytes to mature dendritic cells

doi: 10.1046/j.1365-2249.2001.01672.x

Figure Lengend Snippet: IL-15-induced DC stimulate a strong T-cell response. Allogeneic T cells (2 × 105) from two donors (a) and (b) were cultured separately for 6 d in a 96-well microtiter plate with mature DC (2 × 102−2 × 105 γ-irradiated cells/well) that were generated either by treating with GM-CSF, IL-4 and TNF-α or IL-15. T-cell proliferation was measured by incorporation of [3H]thymidine. DC without T cells, open symbols (□ and ▵); T cells and DC induced with GM-CSF, IL-4 and TNF-α, ▴; T cells and DC induced with IL-15, ▪. Data were expressed as means of triplicate determinations ± s.e.m. Results are representative of six experiments). Statistical analysis in (a) and (b) shows strong dose effect in each group (P < 0·0001 and P < 0·0004), respectively, but pair wise comparison of two sets at each dose shows no significance differences between IL-15 and GM-CSF, IL-4 and TNF-α induced DC. (c). IL-15-induced DC processed and presented recombinant staphylococcal enterotoxin vaccine (rSEB vaccine) and stimulated vaccine-specific T-cell response. CD14+ monocytes (4 × 105) were cultured with IL-15 for 7 d or with a combination of GM-CSF and IL-4 for 6 d followed by TNF-α for 24 h in the presence or absence of different concentrations of rSEB vaccine (0·03 µm to 3 µm) in a 96-well microtitre plate. After 7 days of culture media were removed, and the wells were washed gently to remove residual cytokines. Cells were cultured with autologous (memory and naïve) T-cells (4 × 105). After day 6 of culture, cells were pulsed with [3H]thymidine for 12 h and proliferation was measured by incorporation of [3H]thymidine. Combination treatment with GM-CSF, IL-4 and TNF-α, ▴; IL-15 treatment, ▪; without cytokine treatment (control), U25CF. Data were expressed as means of triplicate determinations ± s.e.m.

Article Snippet: Recombinant cytokines GM-CSF and IL-4 were obtained from Immunex (Seattle, WA), TNF-α from Genzyme Corporation (Cambridge, MA).

Techniques: Cell Culture, Irradiation, Generated, Comparison, Recombinant, Control

Journal:

Article Title: IL-15-induced conversion of monocytes to mature dendritic cells

doi: 10.1046/j.1365-2249.2001.01672.x

Figure Lengend Snippet: Comparison of chemokines secreted by mature dendritic cells

Article Snippet: Recombinant cytokines GM-CSF and IL-4 were obtained from Immunex (Seattle, WA), TNF-α from Genzyme Corporation (Cambridge, MA).

Techniques: Comparison

Journal: PLoS ONE

Article Title: Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

doi: 10.1371/journal.pone.0003381

Figure Lengend Snippet: C57BL/6 and IL-10 −/− were exposed to 75%±2% O 2 from day P7 to P12. Mice were returned to normoxic conditions for 5 days, and on P17 animals were perfused with FITC-dextran, eyes harvested, and retina flatmounts made. Fluorescent microscopy of perfused retinas of (A) normoxic-treated wild-type mice (n = 5), (B) oxygen-treated wild-type mice (n = 5), and (C) oxygen-treated IL-10 −/− mice (n = 4) reveal decreased angiogenesis and increased areas of non-perfusion in IL-10 −/− mice. This experiment was repeated 2 additional times with similar results (D–G) H&E staining of ocular tissue sections from C57BL/6 oxygen-treated mice exhibit extensive preretinal neovascular loops (arrows), whereas (H–K) ocular tissue sections from IL-10 −/− oxygen-treated mice demonstrate a significant reduction in preretinal neovascular loops. Images were acquired at both 40× (D,F,H,J) and 100× (E,G,I,K). (L) The total area of retinal vascularization was quantified using Metamorph™ software as described in , and plotted as a bar graph. IL-10 −/− mice exposed to oxygen had significantly reduced (**P = 0.0006) retinal vascularization compared to wild-type mice exposed to oxygen. (M) A bar graph represents the mean±SD number of neovascular loops of 10 separate sections, with IL-10 −/− mice demonstrating reduced neovascular loops (**P = 0.0071) compared to wild-type mice following OIR.

Article Snippet: Macrophages stimulated with recombinant murine IL-10 (100 ng/ml; R&D Systems) for 10 min were used as a positive control for staining.

Techniques: Microscopy, Staining, Software

Journal: PLoS ONE

Article Title: Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

doi: 10.1371/journal.pone.0003381

Figure Lengend Snippet: (A) The experimental scheme for the treatment of C57BL/6 mice with either rat anti-mouse IL-10 blocking antibodies (n = 4) or rat IgG isotype (n = 6) control antibodies (150 µg) immediately before exposure to oxygen (day P7), immediately after exposure to oxygen (day P12), and 2 timepoints midway through secondary exposure to normoxia (days P14 and P16). On day P17 animals were perfused with FITC-dextran, eyes were harvested, and retinal flatmounts were made. Fluorescent angiography of (B) isotype control treated mice and (C) anti-IL-10 treated mice reveal significantly decreased angiogenesis and increased areas of non-perfusion in anti-IL-10 treated mice following oxygen treatment. (D) The total area of retinal vascularization was quantified using Metamorph™ software as described in and plotted as a bar graph. Anti-IL-10 treated mice had significantly reduced retinal vascularization (**P = 0.0052) compared to IgG treated mice. This experiment was repeated with similar results.

Article Snippet: Macrophages stimulated with recombinant murine IL-10 (100 ng/ml; R&D Systems) for 10 min were used as a positive control for staining.

Techniques: Blocking Assay, Software

Journal: PLoS ONE

Article Title: Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

doi: 10.1371/journal.pone.0003381

Figure Lengend Snippet: Retinal flatmounts of P17, FITC-dextran perfused C57BL/6 normoxia-treated, C57BL/6 oxygen-treated, and IL-10 −/− oxygen-treated retinas were stained with Allophycocyanin (APC) anti-mouse F4/80 antibody. In contrast to the paucity of cells in the (A) normoxic wild-type retina, a significant number of F4/80 + macrophages are observed in the retinas of both (B) C57BL/6 oxygen-treated and (C) IL-10 −/− oxygen-treated mice. (D, E, F) Examining both macrophage infiltrate and blood vessels reveals that APC-labeled macrophages reside primarily along FITC-dextran perfused blood vessels. Since IL-10 did not prevent macrophage infiltration into the retina, we examined the genetic profile of retinal macrophages. Using splenic wild-type macrophages as a baseline, retinal macrophages from wild-type mice exhibit significantly (p<0.05) increased expression of the pro-angiogenic gene (G) nitric oxide compared to IL-10 −/− retinal macrophages following OIR, suggesting that IL-10 promotes angiogenesis by polarizing macrophages towards a pro-angiogenic phenotype.

Article Snippet: Macrophages stimulated with recombinant murine IL-10 (100 ng/ml; R&D Systems) for 10 min were used as a positive control for staining.

Techniques: Staining, Labeling, Expressing

Journal: PLoS ONE

Article Title: Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

doi: 10.1371/journal.pone.0003381

Figure Lengend Snippet: Splenic F4/80 + macrophages from IL-10 −/− mice (A) significantly inhibit (*P = 0.0163) the proliferation of HMVECs following exposure to hypoxia compared to hypoxia-treated wild-type macrophages. Splenic F4/80 + macrophages from wild-type mice do not significantly (P>0.05) inhibit HMVEC proliferation compared to untreated HMVECs. This could be due to (B) increased expression of VEGF in wild-type macrophages compared to IL-10 −/− macrophages. The IL-10 signaling pathway appears to be activated following exposure to hypoxia. RAW 264.7 murine macrophages (C) do not exhibit increased levels of phosphorylation of STAT3 under normoxic conditions (open histogram) compared to IgG stained cells (shaded histogram). However, (E) exposure to hypoxia (open histogram) results in a significant increase in phosphorylation of STAT3 compared to normoxia-treated cells (shaded histogram). This increase in phosphorylation of STAT3 is at levels similar to (D) RAW 264.7 cells exposed to recombinant IL-10 protein for 10 min (open histogram) versus normoxic cells (shaded histogram). Inset numbers indicate percentage of positive cells above normoxic controls. Phosphorylation of STAT3 protein following hypoxia in RAW 264.7 cells was also confirmed with (F) western blot analysis. Phosphorylation of STAT3 occurred following exposure to both recombinant IL-10 protein for 10 min and a 24 hour exposure to hypoxia. Probing of total STAT3 protein was used as a loading control. These findings in RAW macrophages were also observed in primary macrophages (G). Wild-type macrophages treated with recombinant IL-10 protein for 10 minutes or hypoxia for 24 hours demonstrated increased phosphorylation of STAT3 compared to baseline normoxic levels. IL-10 −/− macrophages, however, demonstrated decreased levels of pSTAT3 at baseline normoxic levels compared to wild-type normoxic macrophages, and did not upregulate pSTAT3 in response to hypoxia. STAT3 signaling is still intact in IL-10 −/− macrophages, as phosphorylation of STAT3 in increased in IL-10 −/− macrophages following stimulation with recombinant IL-10 protein. This increase in STAT3 phosphorylation may be due to the (H) significantly increased (*P = 0.0123) production of IL-10 protein by RAW 264.7 macrophages following exposure to hypoxia compared to normoxia-treated RAW macrophages.

Article Snippet: Macrophages stimulated with recombinant murine IL-10 (100 ng/ml; R&D Systems) for 10 min were used as a positive control for staining.

Techniques: Expressing, Staining, Recombinant, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 2. C/EBP, NF-B p50, STAT3, c-Rel, and TBP bind the endogenous CRP promoter. Agarose gel of a ChIP assay performed on Hep3B cells treated with cytokines IL-1 and IL-6 for 0–15 h, as described in Materials and Methods. Abs to C/EBP, NF-B p50, STAT3, c-Rel, and TBP were used in the assays with primers flanking the CRP proximal promoter (118 to 115). The mock is a no Ab control. Input is a 1/10 dilution of total chromatin after sonication and preclearing. C/EBP, NF-B p50, and input are shown (top row). STAT3, c-Rel, and mock are shown in the middle, and TBP is shown in the bottom row. Results are representative of four experiments.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Control, Sonication

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 3. C/EBP binds the endogenous CRP promoter in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show C/EBP occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). Results are an average of three to four ex- periments, each done in duplicate. Error bar represents SD. Statistical sig- nificance of each time point compared with basal levels was determined by a one-way ANOVA and is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 4. p50 occupancy of the CRP promoter changes modestly in the presence of cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show NF-B p50 occupancy expressed as fold change after subtraction of mock and nor- malization to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA at p 0.5, but the experiment had insufficient statistical power to reliably calculate p values.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 5. STAT3 occupancy of the CRP promoter rises modestly in response to cytokines. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were deter- mined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal pro- moter (118 to 115), as described in Fig. 2. Data show STAT3 occu- pancy expressed as fold change after subtraction of mock and normaliza- tion to input signal (see Materials and Methods). Results are an average of three to four experiments, each done in duplicate. Error bar represents the SD. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA is defined. , p 0.5.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 6. c-Rel and TBP occupy the CRP promoter in parallel. Real-time PCR of ChIP assays performed on Hep3B cells treated with cytokines IL-1 and IL-6. Zero time values were determined in each case. Subsequently three time courses were followed 30 min–6 h (n 3 assays) (a), 8–16 h (n 4) (b), and 12–36 h (n 4) (c). Abs were used in the assays with primers flanking the CRP proximal promoter (118 to 115), as described in Fig. 2. Data show c-Rel (solid line, Œ) and TBP (dashed line, f) occupancy expressed as fold change after subtraction of mock and normalization to input signal (see Materials and Methods). a–c, Average of three to four experiments, each done in duplicate. Error bar represents SD. d and e, Profiles from individual ChIP experiments of c-Rel and TBP promoter occupancy. Statistical significance of each time point compared with basal levels was determined by a one-way ANOVA. , p 0.5 is defined for c-Rel and , p 0.5 is indicated for TBP.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 7. CRP mRNA accumulates in response to cytokines. a, Rep- resentative agarose gel of RT-PCR performed on Hep3B cells treated with cytokines IL-1 and IL-6 for the indicated times (hours). CRP mRNA levels are shown at top and -actin mRNA levels are shown at bottom. b, Average quantification of band intensity measured using ImageQuant of CRP mRNA normalized to -actin mRNA (n 4 measurements). Error bar represents SD.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Binding of C/EBPbeta to the C-reactive protein (CRP) promoter in Hep3B cells is associated with transcription of CRP mRNA.

doi: 10.4049/jimmunol.181.4.2420

Figure Lengend Snippet: FIGURE 8. Composite graph of transcription factor occupancy on the CRP promoter and CRP mRNA ac- cumulation in response to cytokines. Common time points for transcription factor occupancy from the 12– 36-h data and CRP mRNA accumulation from the 3–24-h data (12, 18, and 24 h) were plotted together. The left y-axis is the fold change above mock for the transcription factor promoter occupancy. The right y- axis is average normalized band intensity for CRP mRNA accumulation. CRP mRNA (dotted dashed gray line F), C/EBP (black line ), STAT3 (light gray line ‚), p50 (light gray line E), c-Rel (short dashed line Œ), and TBP (black dashed line f) are shown.

Article Snippet: Human recombinant cytokines IL-6 (206-IL) and IL-1 (201-LB) were purchased from R&D Systems.

Techniques:

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Gene expression analysis of macrophage polarization markers (M1, Nos2 and Il1b ; M2, Arg1 ) of alginate bead model (AB model)-elicited peritoneal macrophages from wild type mice via qPCR. ( A ) mRNA expression of Nos2 and Arg1 in AB-elicited macrophages adherence-selected from CD11b-enriched peritoneal lavage, as compared to expression in naïve bone marrow-derived macrophages (M0) or following polarization to M1 (IFN-γ for 24 hrs; M1 ctrl) or M2 (IL-4 for 24 hours; M2 ctrl) states. Gapdh expression was used an internal reference gene control. Data shows mean ± standard deviation (n=3). *** p-value < 0.001 compared to M0 control. ( B ) Peritoneal macrophages elicited by the alginate bead model were treated ex vivo with immunomodulatory agents. qPCR analysis of mRNA expression of indicated genes was normalized to β2-microglobulin ( B2m ) expression was determined following 72 hour incubation with M1-inducing stimuli (LPS at 1 ng/mL + IFN-γ at 10 ng/mL) or M2-inducing cytokines (IL-4 at 20 ng/mL + IL-13 at 10 ng/mL). Fold change in gene expression following treatment is depicted as mean ± standard deviation relative to non-treated controls; ***, p-value < 0.001. Results are representative of 3 independent experiments with peritoneal lavage cells pooled from at least 3 mice.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Expressing, Derivative Assay, Standard Deviation, Ex Vivo, Incubation

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Peritoneal lavage cells elicited by alginate-encapsulated SCs from p16 Ink4a/Luc mice were treated ex vivo with immunomodulatory agents for 72 hours. ( A ) p16 Ink4a promoter-driven luciferase activity (black bars) and β-galactosidase activity (via 4-MUG hydrolysis) (gray bars) were measured following treatment with M1- and M2-polarizing stimuli: LPS at 1 ng/mL, IFN-γ at 10 ng/mL, LPS/IFN-γ co-treatment, Poly(I:C) at 10 μg/mL, IFN-α at 100 U/mL, IL-4 at 20 ng/mL, IL-13 at 10 ng/ml, and IL-4/IL-13 co-treatment. Results are shown as the mean ± standard deviation for at least 3 independent experiments with statistical significance between treated and non-treated samples depicted. ( B ) Microphotograph of SAβG-stained adherence-selected macrophages with or without stimulation with LPS (1 ng/mL) for 72 hours (10x objective). ( C ) mRNA expression of p16 Ink4a and β-galactosidase ( Glb1 ) (relative to B2m expression) in macrophages from wild type mice with or without LPS stimulation for 72 hours analyzed via qPCR, as normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). ( D&E ) Kinetics of p16 Ink4a promoter-driven luciferase activity per cell with or without LPS stimulation ( D ) or IL-4 stimulation ( E ), normalized to activity from non-treated cells at time zero. Results are shown as the mean ± standard deviation (n=3). Statistical significance with respect to non-treated control at time zero is indicated. ( F ) Luciferase activity and β-galactosidase activity (via 4-MUG hydrolysis) from proteose peptone-elicited lavage cells following stimulation with IL-4 (20 ng/mL) for 72 hours, normalized to non-treated controls. Results depicted as mean ± standard deviation (n=3). ( G-J ) Dose-dependent response of JAK1/2 inhibitor Ruxolitinib ( G&H ) and STAT6 inhibitor AS1517499 ( I&J ) on luciferase activity ( G&I ) and viability via CyQuant Direct assay ( H&J ) following 72 hours treatment of AB-elicited macrophages in the presence (gray bars) or absence (black bars) of IL-4 (10 ng/mL) stimulation. Results of luciferase activity and viability are representative of two independent experiments, depicted as mean ± standard deviation of data normalized to respective controls lacking inhibitors (with or without IL-4). Luciferase activity and viability are depicted as the percent signal relative to non-treated (NT) controls. Statistical significance between IL-4 stimulated and non-stimulated cells at each concentration of inhibitor is shown. Results are representative of three independent experiments, depicted as mean ± standard deviation. ( K ) Relative luciferase activity per cell following repolarization of AB-elicited macrophages (via adherence-enriched peritoneal lavage) with M1- and M2-inducing agents. Macrophages were left non-treated (NT) or treated with either LPS (1 ng/ml) or IL-4 (20 ng/ml) for 72 hours (days 1-3), as indicated. For each treatment set, samples were collected at 72 hours (no further treatment; days 4-6 = ×). Alternatively, cells were washed and placed in fresh medium (-), medium containing LPS (1 ng/mL) and IFN-γ (10 ng/mL), or medium containing IL-4 (20 ng/mL) and IL-13 (10 ng/mL) and incubated for an additional 72 hours prior to sample collection (as indicated for days 4-6). Luciferase activity is expressed as the percent activity per cell relative to non-treated (NT) controls after the first 72 hours. Results are representative of two independent experiments. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Ex Vivo, Luciferase, Activity Assay, Standard Deviation, Staining, Expressing, CyQUANT Assay, Concentration Assay, Incubation

Journal: Aging (Albany NY)

Article Title: p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

doi: 10.18632/aging.101268

Figure Lengend Snippet: Primary cultures of adipose-derived mesenchymal stromal cells (AdMSC) isolated from p16 Ink4a/Luc mice were irradiated (20Gy) and cultured for 10 days for senescence induction. Mock irradiated cells were passaged and used as a proliferating cell control. Response of senescent and proliferating AdMSCs to immunomodulatory agents were compared to that of peritoneal lavage cells elicited by the alginate bead model. ( A-C ) Characterization of senescent and proliferating AdMSCs. Microphotographs of SAβG-stained cells depicts positive staining of senescent cells, as well as an enlarged and flattened morphology, compared to that of proliferating cell control ( A ). p16Ink4a promoter-driven luciferase activity ( B ) and β-galactosidase activity measured via 4-MUG hydrolysis ( C ) were measured in senescent and proliferating AdMSCs, confirming senescent phenotypes. ( D-K ) Dose-response curves of LPS ( D&E ), Poly(I:C) ( F&G ), IFN-α ( H&I ), and IFNγ (10 ng/mL), IL-4 (20 ng/mL) and IL-13 (10 ng/mL) ( J&K ) on p16 Ink4a promoter-driven luciferase activity (left panels: D,F,H&J ) and β-galactosidase activity measured via 4-MUG hydrolysis (right panels: E,G,I&K) after 72hr treatment. No effect on viability was observed via CyQuant Direct assay (>80% viability). Results are shown as the mean ± standard deviation for at least 3 experiments, with statistical comparison to non-treated controls; *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. nd, not determined.

Article Snippet: Following elicitation of macrophages via the alginate bead model (2-3 weeks post-injection), or 1 mL of 3% proteose peptone (3 days post-injection) (BD Biosciences; Franklin Lakes, NJ), peritoneal lavage cells were collected and 2.5×10 5 to 5.0×10 5 cells per well were plated overnight in a 24-well plate in complete medium, followed by ≤ 72 hour treatments of carrier-free recombinant mouse cytokines IL-4, IL-13, IFNγ (PeproTech; Rocky Hill, NJ) and IFNα (BioLegend; San Diego, CA), LPS (Sigma Aldrich; St Louis, MO), high molecular weight (HMW) Poly(I:C) (InvivoGen; San Diego, CA), JAK1/2 inhibitor Ruxolitinib (ApexBio; Boston, MA) and STAT6 inhibitor AS1517499 (Axon Medchem; Reston, VA).

Techniques: Derivative Assay, Isolation, Irradiation, Cell Culture, Staining, Luciferase, Activity Assay, CyQUANT Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Live attenuated Salmonella displaying HIV-1 10E8 epitope on fimbriae: systemic and mucosal immune responses in BALB/c mice by mucosal administration

doi: 10.1038/srep29556

Figure Lengend Snippet: ( a ) Serum total IgG levels at day 14 after the final 10E8 peptide boost were measured by quantitative ELISA. The correlations between the serum total IgG concentration and total IgG ASC in BM ( b ), and the specific antibody titer ( c ) were shown. ( d ) IgG subclasses at day 14 after the final 10E8 peptide boost were analyzed by quantitative ELISA. ( e ) The Th1/Th2 bias of immunity was shown as the ratio of IgG2a to IgG1 in concentration. ( f ) The levels of Th1 cytokines (IL-10, IFN-γ, TNF-α) and Th2 cytokines (IL-4, IL-6, IL-21) in sera of immunized mice were detected by quantitative ELISA. R, correlation coefficient. Data shown as mean ± SD of 5 mice in each group. The asterisk indicates a significant difference versus PBS control (* P < 0.05).

Article Snippet: Secretory levels of cytokines IL-4, IL-6, IL-10, IL-21, IFN-γ and TNF-α in the mouse sera were quantitatively determined using the ELISA kits (BOSTER, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Control