Similar to a previous study ( 15 ), we used the proliferation of carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR)-labeled CD4+CD25-T cells (T convs ) to measure the regulatory activity of T regs . T regs were isolated from mice suffering from sepsis as described previously. T convs were isolated from the spleen of C57BL/6 mice with the cell sorter (FACSAria IIu) and labeled with CFSE. T convs (1 × 10 5 ) were cocultured with T regs in a 1:1 ratio. T conv proliferation was induced with anti-CD3 monoclonal antibody (1 μg/mL; BD Biosciences), anti-CD28 (1 μg/mL; BD Biosciences), and IL-2 (5 μg/mL; BD Biosciences) in a 48-well plate for 4 days. After CD4+CD25-gating, the degree of T conv proliferation was examined ( 20 ). In another set of experiments, adenosine ( 21 ) (25 μM; MilliporeSigma) or CGS21680 (1 μM; MedChemExpress) or PBS or adenosine (25 μM; MilliporeSigma) + ZM241385 (1 μM; MedChemExpress), or adenosine (5 μM; MilliporeSigma) + 666-15 ( 22 ) (5 μM; MedChemExpress) was added.
Adenosine
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Sao Tome and Principe, France, Italy, Austria, Canada, Japan, Switzerland
Adenosine is a laboratory chemical used for various research and analytical applications. It is a naturally occurring nucleoside composed of adenine and ribose. Adenosine plays a role in cellular energy transfer and signaling processes. Due to its versatile properties, Adenosine is a widely used compound in many scientific fields.
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Lab products found in correlation
Guanosine, by Merck Group (49 mentions)
Adenosine triphosphate (atp), by Merck Group (41 mentions)
Inosine, by Merck Group (34 mentions)
Uridine, by Merck Group (34 mentions)
Thymidine, by Merck Group (29 mentions)
Cytidine, by Merck Group (28 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (28 mentions)
Adenosine diphosphate (adp), by Merck Group (23 mentions)
Bovine serum albumin (bsa), by Merck Group (23 mentions)
Hemin, by Merck Group (16 mentions)
Adenine, by Merck Group (14 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Hepes, by Merck Group (13 mentions)
Penicillin, by Thermo Fisher Scientific (13 mentions)
L glutamine, by Thermo Fisher Scientific (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Acetylcholine, by Merck Group (10 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (10 mentions)
Cordycepin, by Merck Group (9 mentions)
376 protocols using adenosine
1
Measuring Treg-mediated Suppression of Tconv Proliferation
2
Modulating CTL Perforin Secretion
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CTLs were collected after 72 h of culture with Dynabeads (Thermo Fisher Scientific), which were removed according to the manufacturer’s instructions. CTLs were seeded at a density of 1.5 × 105–2 × 105 cells/well and treated with EVs (4 μg/mL) from vehicle-treated D3H2LN cells (vehicle EVs), EVs (4 μg/mL) from IFN-γ-treated D3H2LN cells (IFN-γ EVs), or 10 μM adenosine (Sigma-Aldrich). To decompose the adenosine, 1 U adenosine deaminase (ADA: Sigma-Aldrich) was added to each well. Perforin protein levels in the supernatant were measured using a Human Perforin ELISA kit (Abcam, Cambridge, UK).
3
Local Delivery of Adenosine and Inhibitors
4
Quantification of OCT3 Protein in Adipose Tissue
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Adipocytes and SVF were isolated from SAT (n = 3) for analysis of OCT3 protein, as described previously (13 (link), 14 (link)). Adipose tissue (500 mg) was digested using collagenase A (Roche) in a shaking water bath at 105 rpm for 1 hour at 37 °C in Medium 199 (Gibco, Life Technologies) supplemented with 6mM glucose, 4% bovine serum albumin (BSA, Sigma), 150nM adenosine (Sigma), pH = 7.4. The floating adipocytes were washed 4 times in Hank's medium (6 mmol/L glucose, 4% BSA, 0.15 μmol/L adenosine, pH = 7.4) and were separated from the medium by filtration through dinonyl phthalate (Merck, Darmstadt, Germany). The SVF fraction was isolated from the collagenase medium by centrifugation for 3 minutes at 1200 rpm and washed with phosphate-buffered saline (PBS). The adipocytes and SVF were used for immunoblotting analysis of OCT3. Total protein levels of the adipocyte and SVF were measured using the BCA protein assay kit (Pierce, Thermo Scientific) and used to extrapolate the total OCT3 protein levels in the whole tissue sample.
5
Adenosine-Functionalized Cardiac Patch
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Our delivery cardiac patch involved the functionalisation with adenosine of a microstructured bilayer based on PLGA (50:50, 40–75 kDa, Sigma Aldrich, USA) and gelatine from porcine skin (gelatine A, Sigma Aldrich, USA), prepared as already described (Cristallini et al., 2016 ; Khademhosseini et al., 2007 ; Kim et al., 2012 ; Zong et al., 2005 ). The functionalisation method with adenosine (>99%, Sigma Aldrich, USA) was optimised (Figure 1 a) in order to reach an immediate and quantitatively elevated release of adenosine in a very short time, as described in Supporting Information . Identical patches, but without adenosine functionalisation, were also produced (MMP). The patches were dried and sterilised according to a previously optimised method (Rosellini, Cristallini, Barbani, Vozzi, & Giusti, 2009 ).
6
Sphere Formation Assay for Tumor Cells
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Single-cell suspensions from either normal or tumor primary cultures were seeded in poly-(2-hydroxyethyl methacrylate) (P3932-25G, Sigma)-coated dishes at a density of 5 × 103 cells/mL in serum-free MEBM (Lonza) supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 1 U/mL heparin, 2% B27, 20 ng/mL EGF, and 20 ng/mL basic fibroblast growth factor. Sphere formation was assessed 5–7 days after seeding. The SFE was defined as the ratio between the number of spheres and the number of cells seeded. To perform the gene expression analysis, first-generation spheres were dissociated and re-seeded as described above in order to obtain second-generation spheres.
When needed, 20 μg/mL of anti-CD73 antibody 2C5 or the isotype-matched control 1A7, 50 μM adenosine 5-(α,β-methylene)diphosphate (APCP; catalog no. M3763), 100 μM caffeine (catalog no. C0750), or 100 μM adenosine (catalog no. A4036; Sigma-Aldrich) were added to suspension cultures on day 0, 2, and 4 after seeding. Sphere formation was assessed 6 days after seeding.
When needed, 20 μg/mL of anti-CD73 antibody 2C5 or the isotype-matched control 1A7, 50 μM adenosine 5-(α,β-methylene)diphosphate (APCP; catalog no. M3763), 100 μM caffeine (catalog no. C0750), or 100 μM adenosine (catalog no. A4036; Sigma-Aldrich) were added to suspension cultures on day 0, 2, and 4 after seeding. Sphere formation was assessed 6 days after seeding.
7
Adenosine Modulation of HSC Mitochondrial Potential
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To examine the effect of adenosine on ΔΨm in HSCs in vitro, HSCs were cultured under undivided conditions (0.5 ng/ml SCF and 0.5 ng/ml TPO) with 0.1∼10 µM adenosine (Sigma) for 48 h and subjected to JC-1 staining. To investigate the effect on intracellular Ca2+ level, immediately after Fluo-4–labeled HSCs were stimulated with 1 µM adenosine, the fluorescent intensity of Fluo-4 was examined by flow cytometry. To examine the role of adenosine A2 receptors in ΔΨm or intracellular Ca2+ level under divided conditions (50 ng/ml SCF and 50 ng/ml TPO), HSCs were treated with 50 µM CV1808 (R&D Systems) for 48 h and subjected to JC-1 or Fluo-4 staining. For in vivo study, 3 mg/kg CV1808 were administrated via i.v. two or three times every 24 h from 1 d after 5-FU treatment, and subsequently ΔΨm or EdC uptake in HSCs were examined at 3 or 4 d after 5-FU administration, respectively. Moreover, both 6 mg/kg SCH442416 and 6 mg/kg PSB1115 were administrated via i.p. four times every 24 h from 3 d after 5-FU treatment. These treated mice were analyzed at 7 d after 5-FU administration.
8
Lymphatic Endothelial Cell Culture
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Human adult dermal microvascular lymphatic endothelial cells (LEC) were purchased from Lonza (HMVEC-dLy; Braine-l′Alleud, Belgium) and used at passages 3 to 5. Cells were cultured at 37°C in a 5% CO2 atmosphere in endothelial growth microvascular (EGM2-MV) medium (Lonza) composed of EBM2 medium with 5% FBS, hydrocortisone, h-FGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF and GA 1000 [9] (link). For drug treatments, cells were washed and cultured in EGM2-MV medium supplemented with 2% FBS, and half of the medium was renewed every 24 h. Adenosine (Sigma-Aldrich) was used at concentration ranging from 0.1 μM to 10 μM and the Adenosine deaminase inhibitor EHNA (Sigma) was used at a concentration of 10 μM to increase Adenosine half-life. CGS21680 and NECA (Sigma-Aldrich) were used as preferential A2a and A2b agonists, respectively.
Primary macrophages were isolated from peritoneal lavage of C57BL/6 mice intraperitoneally injected with 4% thioglycollate (Sigma-Aldrich, St. Louis, MO) 5 days earlier. After washing off non-adherent cells, macrophages were cultured in serum-free medium (RPMI-1640, Lonza) and conditioned medium was collected.
Primary macrophages were isolated from peritoneal lavage of C57BL/6 mice intraperitoneally injected with 4% thioglycollate (Sigma-Aldrich, St. Louis, MO) 5 days earlier. After washing off non-adherent cells, macrophages were cultured in serum-free medium (RPMI-1640, Lonza) and conditioned medium was collected.
9
Characterization of P2Y Receptor Signaling
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All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), including ATP, UTP, ADP, UDP, αβ-meATP, ATPγS, 2meATP, Adenosine, Suramin, RPMI 1640 culture medium and Adenosine. All antibodies were polyclonal and purchased from Alomone Labs, Israel. All selective P2Y receptor used were obtained from Tocris Bioscience, Bristol, United Kingdom. The reagents were stored according manufacturer’s instructions.
10
Visualizing Sec Body Formation
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WT S2 cells (1.5x10 6 ) were plated on coverslips and permeabilized with 10 µg/ml digitonin (Sigma-Aldrich) in KRB for 2h at 26°C. Subsequently, cells were fixed and Sec bodies were visualized by immunostaining of Sec16. To test the effect of ATP, AMP or adenosine on Sec body formation, the semi-intact cells were incubated in the presence of 0.5mM ATP (Sigma-Aldrich), 0.5mM AMP (Sigma-Aldrich) or 0.5mM adenosine (Sigma-Aldrich). Note that the digitonin was not removed. The permeabilization efficiency was determined by using the non-membrane-permeable dye TO-PRO-3 iodide (ThermoFisher) (Figure 7A). The import buffer used in the semi-intact cell system comprised out of 20mM HEPES, 110mM KAc, 2mM MgAc, 5mM NaAc and 0.5mM EGTA (pH was set with KOH).
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