Human breast milk–derived Lactobacillus reuteri DSM17938 (L. reuteri) was provided by BioGaia AB. Lactobacillus acidophilus DDS (La DDS) was provided by D.R. Mack (Children’s Hospital of Eastern Ontario, Ontario, Canada). L. reuteri was prepared as previously described (Liu et al., 2014 (link)). In brief, L. reuteri was anaerobic cultured in deMan-Rogosa-Sharpe (MRS) medium at 37°C for 24 h, and then plated in MRS agar at specific serial dilution and grown anaerobically at 37°C for 48–72 h. Quantitative analysis of bacteria in culture media was performed by comparing absorbance at 600 nm of cultures at known concentrations, using a standard curve of bacterial CFU/ml grown on MRS agar. Each male mouse was given either MRS as control, or L. reuteri (WTL or SFL) or La DDS (WTDDS or SFDDS) in cultured media (107 CFU/day) by gavage, daily, starting from 8 d of age (d8; early treatment) or d15 (late treatment), until the date as indicated in Fig. 3 A and Fig. S2 A, respectively. For treatment with L. reuteri in combination with A2A receptor antagonist SCH58261 (Sigma-Aldrich) to examine the immunological biomarkers, each SF mouse was orally administered 107 CFU of L. reuteri and i.p. injected 2 mg/kg of SCH58261 daily (SFLS) or was i.p. injected 2 mg/kg of SCH58261 daily (SFS) from d8 to the date as indicated in Fig. 3 A , Fig. S2 A, and Fig. 7 A , respectively.
Sch58261
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
SCH58261 is a laboratory product manufactured by Merck Group. It is a chemical compound with the primary function of serving as a research tool for experimental purposes. The detailed specifications and intended applications of this product are not available in this factual, unbiased response.
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Lab products found in correlation
Mrs1754, by Merck Group (6 mentions)
Cgs 21680, by Merck Group (6 mentions)
Dpcpx, by Merck Group (5 mentions)
Adenosine, by Merck Group (5 mentions)
Caffeine, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Sch58261, by Bio-Techne (2 mentions)
Cgs 21680 hydrochloride, by Bio-Techne (2 mentions)
Trypsin inhibitor, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Substance p, by Merck Group (2 mentions)
Ppads, by Merck Group (2 mentions)
Cvt6883, by Merck Group (2 mentions)
Adenosine deaminase ada u46619 9 11 dideoxy 11α 9α epoxymethanoprostaglandin f2α, by Merck Group (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
L name, by Merck Group (2 mentions)
8 cyclopentyl 1 3 dipropylxanthine dpcpx, by Merck Group (2 mentions)
38 protocols using sch58261
1
Probiotic Lactobacillus Strains in Mouse Models
2
Effect of Adenosine Receptor Antagonists on Sleep-Deprived Rats
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To evaluate the effect of an unselective adenosine receptor antagonist in sleep-restricted rats caffeine (Sigma C0750) was used; caffeine was dissolved in saline solution and administrated ip at a unique dose of 0.3mg/kg of body weight at the end of the sleep loss period in the 10th day of sleep restriction. To evaluate the effect of a selective A2A receptor antagonist in sleep restricted rats, SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)-pyrimidine) (Sigma S4568) was used. SCH58261 is a potent A2A adenosine receptor antagonist with good selectivity (48–1561 fold) over A1, A2B, and A3 adenosine receptors, and rapid bioavailability after ip administration [15 (link), 16 (link)]. SCH58261 was dissolved in dimethyl sulfoxide (DMSO) and administrated ip at 0.01, 0.1, or 0.5mg/kg of body weight at the end of the sleep loss period in the 10th day of sleep restriction (3 times each 30 minutes). Rats remained in the acrylic tank during the drug administration to prevent sleep and were sacrificed 30 minutes after the last SCH58261 administration.
3
Investigating Cellular Mechanisms via Pharmacological Inhibition
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NECA and the A2aR antagonist SCH 58261 were obtained from Sigma (St. Louis, MO). The mTOR antagonist OSI-027 was purchased from MedChem Express (Monmouth Junction, NJ). Purified anti-rabbit A2bR polyclonal antibody was purchased from Bioss Antibodies (Edinburgh, UK). Purified anti-rabbit MMP-2, MMP-7, MMP-9, MMP-13, phosphorylated (p)-mTOR, p-PI3K, p-AKT, A2aR, and FITC-IgG (fluorescein isothiocyanate–immunoglobulin G) fluorescent secondary antibody were purchased from Abcam (Cambridge, UK). The EMT Antibody Sampler Kit was purchased from Cell Signaling Technology (Danvers, MA).
4
Adenosine Receptor Antagonist Assay
5
Immunomodulation Pathway Evaluation
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Dopamine, SCH58261, Collagenase A, 2′,7′‐Dichlorofluorescein diacetate (H2DCF), and Coformycin (Erythro‑9‑(2‑hydroxy‑3‑nonyl) adenine) were purchased from Sigma‐Aldrich. 4‐bromomethyl phenylboronic acid (BPBA), Amino polyethylene glycol (PEG), Rhodamine B isothiocyanate (Rhodamine B), and Fluorescein5(6)‐isothiocyanate (FITC) were acquired from Aladdin Industrial Corporation. Bodipy and Live/Dead staining kits were obtained from Thermo Fisher Scientific. Adenosine, CCK‐8, FITC‐Annexin V Apoptosis Detection Kit, Enhanced ATP Assay Kit, BCA assay kit, and Protease inhibitor cocktail for general use (100X) were purchased from Beyotime Biotechnology. Percp‐CRT antibody was purchased from StressMarq Biosciences Inc. Antibodies of APC‐CD73, Alexa Fluor 488‐HMGB1, CD16/32, Foxp3, APC‐CD11c, FITC‐CD80, PE‐CD86, PE‐CD40, FITC‐MHCII, FITC‐CCR7, PE‐CD45, PEcy7‐Gr‐1, APC‐CD11b, APC‐CD3, FITC‐CD4, PEcy7‐CD8α, and PE‐IFNγ were purchased from BioLegend. The antibody of A2AR was obtained from Proteintech Group, Inc. Adenosine ELISA Kit was obtained from Shanghai Jianglai Biological Technology Co., Ltd. ELISA kits of TNF‐α, IL‐6, IFN‐γ, and IL‐12p40 were purchased from Dakewe Biotech Co., Ltd. Cytokines of GM‐CSF, IL‐4, and IL‐2 were purchased from Shanghai Jinan Technology Co., Ltd. DNase I and Bouin's solution were purchased from Beijing solarbio science&technology co., ltd.
6
Adenosine and Chemotaxis Modulation
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Isolated neutrophils were incubated with freshly prepared adenosine (Sigma-Aldrich, UK) at a range of concentrations from 10− 11 M to 10− 5 M immediately prior to studying chemotaxis. Further experiments were performed following preincubation with adenosine receptor antagonists (10− 7 M) DPCPX (A1 antagonist; Sigma-Aldrich, UK), SCH58261 (A2A antagonist; Sigma-Aldrich, UK) or MRS 1334 (A3 antagonist; Tocris Bioscience, UK) for 20 min (37 °C, 5% CO2).
In some experiments neutrophils, either in the presence or in the absence of erythrocytes and/or adenosine (10− 8 M or 10− 5 M), were incubated with 10− 5 or 10− 6 M of either cangrelor (gift from The Medicines Company, New Jersey, USA), ticagrelor (Sequoia Research Products Limited, UK) or dipyridamole (Sigma-Aldrich, UK) for 20 min prior to the chemotaxis assay.
In some experiments neutrophils, either in the presence or in the absence of erythrocytes and/or adenosine (10− 8 M or 10− 5 M), were incubated with 10− 5 or 10− 6 M of either cangrelor (gift from The Medicines Company, New Jersey, USA), ticagrelor (Sequoia Research Products Limited, UK) or dipyridamole (Sigma-Aldrich, UK) for 20 min prior to the chemotaxis assay.
7
Modulation of NK Cell Cytotoxicity
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Cytotoxic activity of NK cells was determined using a Lactate Dehydrogenase (LDH) Detection Kit (Dojindo, Japan). Briefly, the target tumor cells (2.5 × 104/well) were co-cultured with NK-92MI cells in 96-well microplates at different effector-to-target (E: T) ratios for 6 h. In experiments exploring the effect of ATP and Ado analogs on the cytotoxicity of NK cells, NK-92MI cells were pre-treated with ATP-gamma-S (ATP-γ-S, Abcam, USA) or 2-Chloroadenosine (CADO, Abcam) for 3 h before co-culture. In P2Rs and P1Rs blocking experiments, NK-92MI cells were pre-inoculated with 10 μM P2Y11R antagonist NF340 (Santa Cruz Biotechnology, USA) and/or 1 μM A2AR antagonist SCH58261 (Sigma Aldrich) for 30 min before ATP-γ-S or CADO treatment. Anti-DNAM-1 antibody (Ab) mediated blocking assays were performed by incubating NK-92MI cells with anti-human DNAM-1 Ab (clone 102511, R&D Systems, USA) at a final concentration of 10 μg/mL for 1 h before cytotoxicity assay. After 6 h of co-culture, the LDH released in the supernatant was detected and calculated as the manufacturer’s instructions.
8
Intrahepatic and Subcutaneous Tumor Induction
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Intrahepatic tumor injection was performed as described previously.27 (link) In brief, 2.0x105 cells were resuspended in 20 ul of PBS mixed with Matrigel (Corning) and injected into the liver. A subcutaneous tumor was induced by injecting 1.0x106 cells into the left flank of each mouse as previously described.23 (link) Treatment was initiated 5 days post tumor cell injection. The following antibodies were injected intraperitoneally into mice at a dose of 200 μg/mouse at the days indicated in the figures; anti-PD1 (clone 29F.1A12, BioXCell), anti-CD4 (clone GK1.5; BioXCell), and anti-CD8 (clone 2.43; BioXCell). The A2aR inhibitor (SCH58261, Sigma) was administered intraperitoneally at a concentration of 1 mg/kg body weight as indicated. All experiments were conducted according to local institutional guidelines and approved by the NIH Animal Care and Use Committee in Bethesda, MD.
9
Pharmacological Agents Preparation
10
Adenosine Receptor Pharmacology Protocol
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N6-cyclopenthyladenosine (CPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 3-{4-[2-({6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-9H-purin-2-yl}amino)ethyl]phenyl}-propanoic acid (CGS21680) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261) were from Tocris (Bristol, UK). Adenosine and adenosine deaminase (ADA) were from Sigma (St. Louis, MO, USA). CPA, CGS21680, DPCPX and SCH58261 were made up as 5 mM stock solutions in dimethylsulfoxide (Sigma) and dilutions were prepared in artificial cerebrospinal fluid (ACSF, see constitution below), controlling for the impact of the residual amount of dimethylsulfoxide. ADA, DPCPX, SCH58261 and CGS21680 were used in supramaximal but selective concentrations: respectively, 2 U/mL [35 ], 50–100 nM [6 (link)], 50 nM [22 (link),36 (link)], and 30 nM [37 (link)].
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