Exogenous atp

Manufactured by Merck Group

Sourced in Canada

Exogenous ATP is a laboratory reagent that provides an external source of adenosine triphosphate (ATP). ATP is a fundamental energy-carrying molecule found in all living cells and is essential for various cellular processes. Exogenous ATP can be used as a supplement or additive in experimental setups to study ATP-dependent mechanisms or to stimulate cellular responses in vitro.

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Lab products found in correlation

Cd4 t cell isolation kit, by STEMCELL (1 mentions) Atplite luminescence assay system, by PerkinElmer (1 mentions) Infinite 200 pro, by Tecan (1 mentions) Sr12813, by Merck Group (1 mentions) Rifaximin, by Merck Group (1 mentions) Pregnenolone 16α carbonitrile pcn, by Merck Group (1 mentions) Atp bioluminescent assay kit, by Thermo Fisher Scientific (1 mentions) Synergy htx multi mode reader, by Synergy Software (1 mentions) Synergy htx multi mode reader, by Agilent Technologies (1 mentions) Enliten rluciferase luciferin reagent, by Promega (1 mentions)

5 protocols using exogenous atp

CD4+ T Cell Activation by HNSCC Exosomes

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Exosomes of HNSCC patients were co-incubated with healthy human CD4(+) T cells (10⁵/100 µL) in the presence of 20 μM of exogenous ATP (Sigma Life Science). Exosome samples used were derived from the total, CD45(+), or CD45(−) fraction. The CD4(+) T cells were isolated using a CD4+ T cell isolation kit (17952, Stemcell Technologies) according to manufacturer’s instruction. Control samples incubated with no exosomes (PBS only) and without ATP were used.
After 20h of incubation, percentages of CD4+CD39+ T cells were determined by flow cytometry using anti-CD4-FITC Ab (11-0049-42, eBioscience) and anti-CD39-PECy7 Ab (25-0399-42, eBioscience, San Diego, CA, USA). The protocol was previously described in detail [8 (link),20 (link),31 (link)].

Beccard I.J., Hofmann L., Schroeder J.C., Ludwig S., Laban S., Brunner C., Lotfi R., Hoffmann T.K., Jackson E.K., Schuler P.J, & Theodoraki M.N. (2020). Immune Suppressive Effects of Plasma-Derived Exosome Populations in Head and Neck Cancer. Cancers, 12(7), 1997.

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ATP-Induced Luminescence in B Cells

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CD19⁺ B cells (3 × 10⁴ per well, viable cells counted in a trypan blue dye) obtained from NC, after seven days of chemotherapeutic treatment, were incubated with 20 µM exogenous ATP (Sigma-Aldrich, St. Louis, MO) in 96-well plates for 90 min. The concentration of nonhydrolyzed ATP was determined by measuring the frequency of luminescent events in a luciferase-based detection system (ATPlite Luminescence Assay System, PerkinElmer, Waltham, MA) using a luminescence reader (Infinite^® 200 Pro, Tecan, Maennedorf, Switzerland). Control samples included wells with ‘no cells’ or ‘no ATP’.

Ziebart A., Huber U., Jeske S., Laban S., Doescher J., Hoffmann T.K., Brunner C., Jackson E.K, & Schuler P.J. (2017). The influence of chemotherapy on adenosine-producing B cells in patients with head and neck squamous cell carcinoma. Oncotarget, 9(5), 5834-5847.

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PXR Agonist Activation of Macrophages

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For experiments in mouse macrophages, the rodent selective PXR agonist pregnenolone 16α-carbonitrile (PCN; Sigma-Aldrich Canada, Oakville, Ontario, Canada) was prepared, as described above. For experiments in human macrophages, the human-selective PXR agonists rifaximin and SR12813 (Sigma-Aldrich Canada) were dissolved in sterile dimethylsulfoxide and added to culture media to attain the final experimental concentrations. As a vehicle control, identical volumes of dimethylsulfoxide were added and did not exceed a concentration of 1% v/v in media. The addition of exogenous ATP (5 mM; Sigma-Aldrich Canada) was used as a positive control in all experiments assessing inflammasome activation, as we have done previously (Ng et al., 2010 (link); Hirota et al., 2011 (link)).

Hudson G., Flannigan K.L., Venu V.K., Alston L., Sandall C.F., MacDonald J.A., Muruve D.A., Chang T.K., Mani S, & Hirota S.A. (2019). Pregnane X Receptor Activation Triggers Rapid ATP Release in Primed Macrophages That Mediates NLRP3 Inflammasome Activation. The Journal of Pharmacology and Experimental Therapeutics, 370(1), 44-53.

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Measuring ATP Hydrolysis in T Cell Subsets

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Sorted CD39⁺ Tconv, CD39⁻ Tconv and Treg cells from B16F10-OVA tumor-bearing Foxp3-EGFP mice were incubated with 5 µM of exogenous ATP (Sigma-Aldrich) as previously described.^{19 (link)} Cell-free medium was then analyzed for residual ATP with ATP bioluminescent assay kit (INVITROGEN). Bioluminescent activity was measured with a Synergy HTX Multi-Mode Reader.

Bossio S.N., Abrate C., Tosello Boari J., Rodriguez C., Canale F.P., Ramello M.C., Brunotto V., Richer W., Rocha D., Sedlik C., Vincent-Salomon A., Borcoman E., Del Castillo A., Gruppi A., Fernandez E., Acosta Rodríguez E.V., Piaggio E, & Montes C.L. (2023). CD39+ conventional CD4+ T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade. Oncoimmunology, 12(1), 2246319.

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CD39 Activity Evaluation in T Cells

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For CD39 activity evaluation, 5x10 4 sorted cells were cultured for 10 min in phenol red-free RPMI-1640 with 20 µM of exogenous ATP (SIGMA). Cell-free medium with ATP alone was used as control.
For ATP hydrolysis inhibition cells were incubated for 16 h in presence or absence of 250 µM ARL67156 and ATP was added in the last 10 min. Cell-free medium was then analyzed for remaining eATP with ENLITEN rLuciferase/Luciferin reagent (Promega). Bioluminescent activity was measured with a Synergy HTX Multi-Mode Reader (BioTek). The percentage of ATP hydrolysis was calculated as previously described (18) . Spleen CD8 + T cells were magnetically purified from tumor-free mice.

Canale F.P., Ramello M.C., Núñez N., Araujo Furlan C.L., Bossio S.N., Gorosito Serrán M., Tosello Boari J., Del Castillo A., Ledesma M., Sedlik C., Piaggio E., Gruppi A., Acosta Rodríguez E.A, & Montes C.L. (2018). CD39 Expression Defines Cell Exhaustion in Tumor-Infiltrating CD8⁺ T Cells. Cancer research, 78(1).

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