The adenosine pathway was inhibited using the following agents. The A2aR inhibitor vipadenant (provided by Juno Therapeutics, a Celgene company) was suspended in 40% Captisol (Captisol Ligand Technology) and 10% (w/s) PEG400 (MilliporeSigma) in PBS, and SCH58261 (MilliporeSigma) was dissolved in DMSO (MilliporeSigma) and then diluted in Cremophor EL (MilliporeSigma) and 0.9% NaCl (final concentration, 15% DMSO and 15% Cremophor EL). These agents have been reported to have blood-brain barrier penetration in human subjects with Alzheimer’s and Parkinson’s disease (16 (link), 22 (link)–27 (link)). The mice were treated daily for 21 days, starting on day 3 after glioma implantation, via i.p. injection with either SCH58261 (10 mg/kg) or with vipadenant (60 mg/kg). The CD39 inhibitor POM-1 (Tocris) was dosed on the same schedule at 5 mg/kg. Anti-CD73 (clone TY/23; Bio X Cell; RRID: AB_10950310) or IgG control (clone 2A3, Bio X Cell; RRID: AB_1107769) was administered intravenously at a dose of 200 μg/mouse on days 3, 6, 10, 14, 17, and 21. To evaluate complementary immune suppression blockade, an anti PD-1 (clone RMP1-14; Bio X Cell; RRID: AB_10949053) or IgG control (clone 2A3, Bio X Cell; RRID: AB_1107769) was administered i.p. at a dose of 200 μg/mouse on days 7, 9, and 10.
Pom 1
Manufactured by Bio-Techne
Sourced in United States
The POM-1 is a laboratory automation system designed for high-throughput sample processing. It provides precise pipetting, sample mixing, and incubation capabilities to streamline workflows and improve efficiency in research and diagnostic laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine, by Merck Group (4 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Arl 67156 trisodium salt, by Bio-Techne (2 mentions)
Sch442416, by Bio-Techne (2 mentions)
Compound c, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Apyrase, by Merck Group (2 mentions)
Cgs 21680, by Merck Group (2 mentions)
Atpγs, by Merck Group (2 mentions)
Recombinant il 23, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Adenosine triphosphate (atp), by Bio-Techne (2 mentions)
Milli q biocel, by Merck Group (2 mentions)
Clone rmp1 14, by BioXCell (1 mentions)
Clone 2a3, by BioXCell (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cremophor el, by Merck Group (1 mentions)
17 protocols using pom 1
1
Modulating Adenosine Signaling in Glioma
2
Modulating CD39 ATPase and Adenosine Signaling
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To inhibit CD39 ATPase activity, 100–400 μM of the nondiffusible ATPase inhibitor ARL 67156 trisodium salt (Tocris) was added to cultures on day 1 after anti-CD3/CD28 Abs activation. Functional assays (cytokine production, T/B cell coculture), apoptosis assays, and flow cytometry for pAMPK on CD39+ T cells were performed on day 4. Alternatively, ATPase activity was inhibited in day 4 activated CD4+ T cells with 10 or 50 μM of the ENTPD inhibitor POM-1 (TOCRIS) for 6 hr before assaying apoptosis.
To block Adenosine stimulation of the A2A receptor, SCH 442416 (Tocris) was added at a concentration of 10 μM on day 1 after initial stimulation.
To inhibit pAMPK activity, 2 μM Compound C (Sigma-Aldrich) or control DMSO was added to CD4+ T cell cultures on day 1 after stimulation with anti-CD3/CD28 beads. pAMPK, p-p53 (ser15), and p21 expression in CD39+ T cells was determined by western blotting; apoptosis was determined by flow cytometry.
Adenosine (0.5, 1.0, or 1.5 mM; Sigma-Aldrich) was added to CD4+ T cell cultures on day 3 after stimulation. Phosphorylation of AMPK in purified treated and untreated CD39− CD4 T cells was examined by western blotting on day 4.
To block Adenosine stimulation of the A2A receptor, SCH 442416 (Tocris) was added at a concentration of 10 μM on day 1 after initial stimulation.
To inhibit pAMPK activity, 2 μM Compound C (Sigma-Aldrich) or control DMSO was added to CD4+ T cell cultures on day 1 after stimulation with anti-CD3/CD28 beads. pAMPK, p-p53 (ser15), and p21 expression in CD39+ T cells was determined by western blotting; apoptosis was determined by flow cytometry.
Adenosine (0.5, 1.0, or 1.5 mM; Sigma-Aldrich) was added to CD4+ T cell cultures on day 3 after stimulation. Phosphorylation of AMPK in purified treated and untreated CD39− CD4 T cells was examined by western blotting on day 4.
3
Modulation of Intestinal and Spleen Cell Responses
4
Ectonucleotidase Activity Assay for Mononuclear Cells
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Ectonucleotidase assays were performed using peritoneal mononuclear cells from WT (and mutant) mice. These cells were seeded in a 96-well plate (2 × 105 cells/200 μL/well), incubated for 3 hours, and washed twice to remove unattached cells. Then, fresh medium was added and cells were incubated in 100 μL of medium with antibodies (CTRL-m1, WT 5F2-m1, WT 5F2-m2c, and Afuc 5F2-m2c αmCD39 antibodies) for 24 hours or 26 μM POM-1 (TOCRIS) for 30 minutes. For ectonucleotidase analyses, 100 μL of 100 μM ATP (Sigma-Aldrich) was added to each well and incubated for 20 minutes. Supernatants (25 μL) were transferred into a 96-well opaque-walled multiwell plate (BrandTech Scientific) and mixed with 50 μL of CellTiter-Glo Reagent (Promega) for 2 minutes. Samples were incubated for 10 minutes at room temperature in the dark and then luminescence was read using a Synergy Neo2 Multi-Mode Reader.
5
Suppressing GVHD with GMSCs and nTregs
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Mice were irradiated by 2.5 cGy TBI using a 137Cs source 2–4 h before PBMC injection. CD25-depleted PBMC (20 × 106) were injected intravenously into mice via tail vein. Four hours later, PBS, GMSCs, human Fibroblasts, or nTregs were intravenously co-transfused. Mice were monitored every 2–3 days for weight loss and assessed for symptoms of GVHD. Blood samples were taken weekly to detect the percentages of human CD3+ T cells and cytokine production and Foxp3 expression among CD4+ cells. Serum samples were used to measure cytokines and antibodies by ELISA. At the terminal point of observation, mice were sacrificed for cytokine measurements and histologic analysis. To determine the underlying mechanisms, a tryptophan analog 1-Methyl-d -tryptophan (1-MT) (Sigma) was administered i.p. at 10 mg/mouse/day for 14 consecutive days. Additionally, GMSCs cells were pretreated with POM-1 (Tocris Bioscience; 100 µM) overnight before being injected into mice.
6
Isolation and Culture of Intestinal and Spleen Cells
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Intestinal LPLs or spleen cells were isolated from Rag1−/− mice and cultured in completed RPMI 1640 medium supplemented with 10% FBS, 2‐Mercaptoethanol (50 μmol L−1, Gibco, Waltham, MA, USA), l ‐glutamine (2 mmol L−1, Gibco) and antibiotics (Penicillin and Streptomycin, 100 U mL−1, Gibco). When indicated, recombinant IL‐23 (20 ng mL−1, eBioscience, San Diego, CA, USA), POM‐1 (2–50 μmol L−1, Tocris Bioscience), APCP (2–50 μmol L−1), Apyrase (10 μmol L−1, Sigma), ATPγS (5–10 μmol L−1, Sigma), and A2A agonist CGS 21680 (1–10 μmol L−1, Merck Chemicals, Darmstadt, Germany) and their respective vehicle control (i.e. DMSO or dH2O) or combination of these reagents were added into cell cultures. For detecting cytokines in supernatants, cells were cultured for overnight, and for intracellular staining of IL‐22, cells were stimulated for 4 h in the presence of GolgiPlug (BD Bioscience, San Jose, CA, USA). In vitro cell cultures were performed in triplicate and repeated two or more times.
7
Modulating CD39 ATPase and Adenosine Signaling
8
Measuring CD39-mediated ATP Hydrolysis
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Non-stimulated PBMCs were washed three times with phosphate-free buffer (0.5 mM CaCl2, 120 mM NaCl, 5 mM KCl, 50 mM Tris-HCl, pH 8), resuspended in phosphate-free buffer in the presence or absence of 250 µM ATP (Sigma-Aldrich) and then incubated at 37 °C for 30 minutes. The ATP hydrolysis was stopped by placing cells on ice for 10 minutes. Then, 80 µl of the supernatants were used to measure inorganic phosphate using the Malachite Green Phosphate Assay Kit (BioChain), according to the manufacturer’s instructions. To exclude the possibility that non-enzymatic degradation of ATP and the direct release of phosphate by PBMCs contributed to the phosphate levels measured, controls using ATP incubated without cells and cells incubated without ATP were included, respectively. To determine whether the inorganic phosphate was selectively produced by CD39 activity, the NTPDase inhibitor POM-1 (10 µM; Tocris) was used.
9
Mesenchymal Stem Cells Enhance Bone Formation
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At 8 weeks of age, mice were either sham or ovariectomized. Surgically removed ovaries were examined by the histology to verify successful ovariectomy. 2 × 106 GMSCs or human dermal fibroblasts (control cells) were intravenously injected into each mouse on day 14 after surgery. To determine underlying mechanisms, GMSCs were pretreated with CD39 inhibitor POM-1 (Tocris Bioscience; 100 μM) overnight and washed twice with PBS before being injected into mice. Mice were subsequently analyzed at 18 weeks of age for assessment of dynamic bone formation by intraperitoneal calcein injection (10 mg/kg per mouse) at 14 days and 2 days prior to completion of the experiments, which were repeated at least three times. GMSCs that had been used at each time period were obtained from different donors, and mice in each experimental time interval received the same cell population from the same donor.
10
Multiparameter Analysis of DC Activation
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We used: PE-labeled antibodies to CD40 (clone 5C3), CD80 (clone L307.4), CD83 (clone HB15e), CD39 (clone TU66), PDL-1 (clone MIH1), as well as anti-CD4-APC (clone SK3) all from Becton Dickinson: (BD), anti-CD86-PE (clone HA5.2B7, Beckman Coulter), anti-IL-12p35/p70-PE (clone REA121, Miltenyi Biotech), anti-IL-12/IL-23p40-APC (clone C11.5, BioLegend), anti-IL-10-PE (clone JES3-19F1, BD), anti-IFN-γ-APC (clone B27, BD), anti-IL-13-PE (clone JES10-5A2, BD), anti-IDO-1-APC (clone 70083, R&D), anti-HO-1 (HO-1-1, Thermoscientific). For blocking experiments, we used rat neutralizing Ab to IL-10 (10 μg/ml, BD), IL-10R (10 μg/ml, R&D Systems), TLR2, TLR6, and Rat IgG control (20 μg/ml, InvivoGen), the JNK inhibitor SP600125 (10 μM, Abcam), the inhibitor of the CD39 ecto-nucleotidase activity Pom1 (10 μM, Tocris) and the IDO1 inhibitor (400 μM, Sigma Aldrich). DC were incubated with the inhibitor for 45 min before bacteria addition.
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