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Enliten atp assay

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The ENLITEN ATP Assay is a luminescence-based kit used to detect and quantify adenosine triphosphate (ATP) in biological samples. It provides a sensitive and rapid method for measuring ATP levels, which is an indicator of cellular energy status and viability.

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45 protocols using enliten atp assay

1

ATP Release in HMEC Cells

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Concentration of ATP in cell media was detected by luciferin-luciferase assay (ENLITEN ATP Assay, Promega; Charbonnieres, Fr). HMEC were plated at 500 × 103 cells/cm2, growth arrested in FCS-free medium and exposed to apyrase (20 U/ml) or GAP26 (500 μM) or probenecid (100 μM) and/or monocytes stimulated or not by M-CSF (100 nM). Supernatants were collected after 1h to 12h, put on ice and centrifuged at 12,000 g for 10 min.
Thuringer D., Berthenet K., Cronier L., Jego G., Solary E, & Garrido C. (2015). Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells. Oncotarget, 6(12), 10267-10283.
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2

Cellular ATP Quantification in Cancer Cells

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Cellular ATP level was measured in H69 and SW780 cells treated with calcium with or without electroporation using ATP luminescence assay (Enliten ATP Assay; Promega) after cell lysis. For ATP assay 11.5 × 104 cells/ml H69 cells or 4.6 × 104 cells/ml SW780 cells in 100 μl culture medium were in 96-well plates in accordance with initial optimization.
Cells were treated according to the electroporation protocol. Cells electroporated with HEPES buffer, non-electroporated cells with calcium, and untreated cells were used as controls. Cells thrice thawed and frozen, then sonicated (Ultrasonic Bath Branson 1200), were used as control (dead cells). After centrifugation of the 96-well plates supernatant was removed. Cellular ATP content 1, 2, 4, and 8 hours after treatment was determined by lysing the cells (30 min) then adding 100 μl rl/l reagent and measuring light emission using a luminometer (LUMIstar, Ramcon).
Hansen E.L., Sozer E.B., Romeo S., Frandsen S.K., Vernier P.T, & Gehl J. (2015). Dose-Dependent ATP Depletion and Cancer Cell Death following Calcium Electroporation, Relative Effect of Calcium Concentration and Electric Field Strength. PLoS ONE, 10(4), e0122973.
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3

Extracellular ATP Levels in Cancer Cells

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PANC-1, PAN02, and CT26 cells were treated with chemotherapeutic agents or viruses for 24 h, and levels of extracellular ATP were measured using an ENLITEN ATP assay (Promega, Madison, WI, USA).
Hashimoto M., Kuroda S., Kanaya N., Kadowaki D., Yoshida Y., Sakamoto M., Hamada Y., Sugimoto R., Yagi C., Ohtani T., Kumon K., Kakiuchi Y., Yasui K., Kikuchi S., Yoshida R., Tazawa H., Kagawa S., Yagi T., Urata Y, & Fujiwara T. (2024). Long-term activation of anti-tumor immunity in pancreatic cancer by a p53-expressing telomerase-specific oncolytic adenovirus. British Journal of Cancer, 130(7), 1187-1195.
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4

Quantifying Cellular ATP Levels

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Cellular ATP was quantified using the ENLITEN ATP assay, according to the manufacturer's instructions (Promega Ltd, Southampton, UK), and normalized using a bicinchoninic acid protein assay. Briefly, NHU cells were cultured to 80% confluence in 10 cm dishes before exposure to 3 mmol/L ketamine or medium only (KSFMc) for 48 hours. Cultures were harvested, lysed in 2% trichloroacetic acid, and assayed for activation of luciferase against an ATP standard curve in a luminescence plate reader (BMG Labtech Ltd., Aylesbury, UK).
Baker S.C., Shabir S., Georgopoulos N.T, & Southgate J. (2016). Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor–Independent Pathway Characterized by Mitochondrial Stress. The American Journal of Pathology, 186(5), 1267-1277.
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5

Measuring Intracellular ATP Release

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To measure the release of intracellular ATP, cell culture supernatants were collected and the concentrations of ATP were measured using luciferin‐based ENLITEN ATP Assay (Promega) kits in accordance with the manufacturer's protocol.
Meng J., Yang X., Huang J., Tuo Z., Hu Y., Liao Z., Tian Y., Deng S., Deng Y., Zhou Z., Lovell J.F., Jin H., Liu Y, & Yang K. (2023). Ferroptosis‐Enhanced Immunotherapy with an Injectable Dextran‐Chitosan Hydrogel for the Treatment of Malignant Ascites in Hepatocellular Carcinoma. Advanced Science, 10(20), 2300517.
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6

ATP Release Assay in THP-1 Cells

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To study ATP released from THP-1 cells, the supernatant or the cell lysates of CM-treated THP-1 cells were subjected to the luciferin-based ENLITEN ATP Assay (Promega), according to the manufacturer’s instructions.
Liu C., Shen Y., Huang L, & Wang J. (2022). TLR2/caspase-5/Panx1 pathway mediates necrosis-induced NLRP3 inflammasome activation in macrophages during acute kidney injury. Cell Death Discovery, 8, 232.
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7

Extracellular ATP Levels in Treated Cells

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iRTN-1c MCA205 cells were treated (or not) by 1 µM MTX, 150 µM CDDP, 0.3 µM TET, or CDDP+TET for 24 h, then extracellular ATP levels were measured by the luciferin-based ENLITEN® ATP Assay (Promega), in excess of luciferin and luciferase, as indicated by the manufacturer. ATP-driven chemoluminescence was recorded on a Fluostar Multiwell Plate Luminometer (BMG Labtech).
Michaud M., Sukkurwala A.Q., Di Sano F., Zitvogel L., Kepp O, & Kroemer G. (2014). Synthetic induction of immunogenic cell death by genetic stimulation of endoplasmic reticulum stress. Oncoimmunology, 3, e28276.
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8

Fluorescence Assay for ATP Vesicles

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For the detection of ATP containing vesicles, the cells were labeled with quinacrine as described.12 (link) In short, cells were incubated with 5 μm quinacrine and 1 μg/ml Hoechst 33342 in Krebs-Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.7 mM KH2PO4, 2 mM CaCl2, 6 mM glucose and 25 mM Hepes, pH 7.4) for 30 min at 37 °C. Thereafter, cells were rinsed with Krebs-Ringer and living cells were microscopically examined. Alternatively, the concentration of extracellular ATP was assessed by means of the ENLITEN ATP assay (Promega, Madison, WI, USA), based on luciferin conversion, following the manufacturer's instructions. Chemoluminescence was measured using an i3 Paradigm multi-label reader (Molecular Devices).
Zhou H., Forveille S., Sauvat A., Yamazaki T., Senovilla L., Ma Y., Liu P., Yang H., Bezu L., Müller K., Zitvogel L., Rekdal Ø., Kepp O, & Kroemer G. (2016). The oncolytic peptide LTX-315 triggers immunogenic cell death. Cell Death & Disease, 7(3), e2134-.
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9

Cytotoxicity Evaluation: MTT and ATP Assays

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For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromid (MTT) viability assay, cells were seeded in 96-well plates (Sigma, München, Germany). For single treatment in MTT assay, serial dilutions of cytostatic drugs or a dilution of MOIs were generated and added to the different cell lines. MTT was added and after dissolving, the produced purple formazan with sodium dodecyl sulfate (SDS) absorption was measured at 562 nm by spectrophotometer (enzyme-linked immunosorbent assay [ELISA] Reader; Bio-Tek Instruments, Bad Friedrichshall, Germany).
The ATP assay (Enliten® ATP Assay; Promega Corporation, Madison, WI, USA) was performed as described by the manufacturer. Ninety-six-well plates (Greiner Bio-One, Frickenhausen, Germany) were filled with 50 µL of supernatants, which were collected by aspirating medium from the coculture. Luminescence was measured with Apliskan® (Thermo Fisher Scientific, Vantaa, Finland).
Heinrich B., Klein J., Delic M., Goepfert K., Engel V., Geberzahn L., Lusky M., Erbs P., Preville X, & Moehler M. (2017). Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes. OncoTargets and therapy, 10, 2389-2401.
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10

Cytotoxic Stress Biomarker Profiling

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Tumor cells (6 × 104) were adhered in 24-well plates in 1ml of heat-inactivated complete medium and treated with OXP or CDDP for 24 or 48 h. Supernatants were collected, dying floating cells removed by centrifugation and supernatants frozen immediately. ATP secretion was measured with the ENLITEN ATP Assay (Promega, catalog# FF2000) according to the manufacturer's protocol. HMGB1 release was assessed by enzyme-linked immunosorbent assay (ELISA, IBL International, catalog# ADI-SPA-810-D) according to manufacturer's instructions.
Di Blasio S., Wortel I.M., van Bladel D.A., de Vries L.E., Duiveman-de Boer T., Worah K., de Haas N., Buschow S.I., de Vries I.J., Figdor C.G, & Hato S.V. (2016). Human CD1c+ DCs are critical cellular mediators of immune responses induced by immunogenic cell death. Oncoimmunology, 5(8), e1192739.
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