Cells were seeded in 384-well plates (Corning Life Sciences Plastic, Bedford MA, USA, #3570) at the final concentration of 0.02 × 106/mL per condition. Small molecules were added with a nanometric dispenser Tecan D300e (Tecan Trading AG, Switzerland), and cellular viability was assessed after 72 hours of drug treatment using a CellTiter-Glo ATP assay (Promega Corporation, Madison, WI, USA, #G7573). IC50 and the area under the curve (AUC) were calculated using GraphPad Prism software (La Jolla, CA, USA).
Celltiterglo atp assay
Manufactured by Promega
Sourced in United States
CellTiter-Glo is a luminescent cell viability assay that quantifies adenosine triphosphate (ATP) as an indicator of metabolically active cells. The assay reagent lyses cells and generates a luminescent signal proportional to the amount of ATP present, which is directly correlated to the number of viable cells.
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Lab products found in correlation
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Non targeting control sirna, by GenePharma (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Camp glo assay, by Promega (1 mentions)
Amersham ecl prime western blotting reagent, by GE Healthcare (1 mentions)
Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Flt3l, by R&D Systems (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Interleukin 7 (il 7), by R&D Systems (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Spectramax gemini em microplate reader, by Molecular Devices (1 mentions)
Amp glo assay, by Promega (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
16 protocols using celltiterglo atp assay
1
Cell Viability Assessment with Small Molecules
2
Optimized Cell Culture and Reporter Assay Protocol
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Cell culture and growth conditions for HEK 293T and U2OS cells were performed as previously described [12 (link),46 (link),84 (link)]. Briefly, 293T and U2OS cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum, as previously described [84 (link),85 (link)]. For the reporter assay, 293T cells were cultured and seeded in 96-well plates, and transfected with desired plasmids using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 h later and assayed with the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase as an internal control for transfection efficiency, as detailed in our previous studies [37 (link),46 (link),86 (link)]. The luminescent CellTiter-Glo ATP assay (Promega) was used to assess cell viability. For the Lumicycle assay, samples were collected at day 6 at the end of the run when the clock phenotype was at the strongest. For the transient transfection assay, samples were collected 24 h post transfection when the Dual-Glo Luciferase Assay was performed.
3
Assessing Oocyte ATP and cAMP Levels
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Four groups of 30 control or SPAG-1–depleted oocytes were retrieved for ATP and cAMP assessment. ATP and cAMP relative concentrations were evaluated by using the Cell-Titer-Glo ATP Assay (Promega, Madison, WI) and cAMP-Glo Assay (Promega), respectively, according to the manufacturer’s instructions. Of note, the cAMP assay is based on the principle that cAMP stimulates protein kinase A holoenzyme activity, decreasing available ATP and leading to decreased luminescence intensity in a coupled luciferase reaction. Thus, elevated cAMP abundance is indirectly revealed by decreased luminescence intensity of ATP.
4
Mitochondrial Membrane Potential Assay
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TMRM and JC-1 probes were from Invitrogen Life Technologies (Carlsbad, CA, USA and Dun Laoghaire, Ireland). Amersham™ ECL™ Prime Western blotting reagent was from GE Healthcare Life Sciences (Waukesha, WI, USA), pre-made acrylamide gels, running and transfer buffers were from GeneScript (Piscataway, NJ, USA), RIPA buffer, BCA™ Protein Assay kit and Pre-stained Protein Ladder were from Thermo Fisher Scientific (Rockford, IL, USA). CellTiter-Glo® ATP Assay was from Promega (Madison, WI, USA). PhosphoStop Phosphatase Inhibitor and complete Protease Inhibitor Cocktail Tablets were from Roche (Dublin, Ireland). Dulbecco’s Modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) media, cycloheximide and all the other reagents were from Sigma-Aldrich (St. Louis, MO, USA). Primary and secondary antibodies are listed in Additional file 4 . Plastic and glassware were from Sarstedt (Ireland), Corning Life Sciences (Corning, NY), Greiner Bio One (Frickenhausen, Germany) and Pecon (Erbach, Germany).
5
Leukemia Cell Viability Assay
6
Measuring Intracellular AMP and ATP
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Intracellular AMP and ATP levels were measured using AMP-Glo™ Assay (Promega) and CellTiterGlo™ ATP assay (Promega) according to the manufacturer’s instruction.
7
Modulation of Cell Metabolism and Apoptosis
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C43 (10 µM), AC220 (1 µM), or A70 (1 µM) were used unless otherwise stated. STS (1 µM), MG132 (10 µM), ClQ (50 µM), LY294002 (10 µM), GDC-0941 (0.5 µM), MK2206 (1 µM), oligomycin (0.1 µM), Rotenone (0.1 µM), Etomoxir (5 µM), T0070907 (5 µM), d -glucose (11 mM), 2DG (1, 2 or 5 mM), zVAD.fmk (20 µM), 7N-1 (20 µM), and doxorubicin (1.25 µg/ml) were also used. DMSO (0.1%) was used as a control. Cell viability and ATP levels were measured using a CellTiterGlo ATP assay (Promega). The ADP/ATP and NAD+/NAHD ratios were determined with assay kits (Abcam) according to the manufacturer’s instructions. Annexin V/PI staining was detected by using a flow cytometer using an FITC Annexin V Apoptosis Detection kit I (BD). Cell death/survival was measured using the ToxiLight bioassay (Thermo Fisher Scientific). Cellular fractionation was performed using differential centrifugation and hypotonic lysis of lysosomes, according to Schröter et al. (1999) (link). Coimmunoprecipitation was performed using RIPA buffer and 1 µg antibody per sample incubated with A/G for 2 h. For genetic knockdown experiments, scramble (SCR)/nontargeting (N.T) or siRNA targeting ATG7 (GE Healthcare) Lamp2A, Hsc70 (GE Healthcare), FLT3 (Santa Cruz Biotechnology, Inc.), or HK2, p53, SGT1, and FAM3C (GenePharma) were used at 40 nM. siRNA efficiency was monitored 48–72 h after transfection.
8
Cell Viability via ATP Assay
9
Photothermal Therapy Nanoparticle Viability
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The viability of INAPs-treated SM1 or B16F10 (1 × 106 cells) was determined at varying nanoparticle concentrations (0.5–2.0 mg/mL) in the presence or absence of an NIR laser by suspending cells in PBS (200 µL). Post-PTT, the cells were then transferred to 6-well plates and incubated in media for 24 h. The cells were then collected, suspended in 400 µL of PBS, and assessed for viability in triplicate using the Cell Titer-Glo ATP assay (following the manufacturer’s instructions) (Promega). As controls, ICG-PLGA at 0.5 to 2.0 mg/mL and free NextA (5 µM) were used. Luminescence was measured using a SpectraMax i3x Multi-mode microplate reader from Molecular Devices, LLC (San Jose, CA, USA).
10
Assessing Chromium Cytotoxicity using ATP Assay
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A Promega CellTiter-Glo® ATP assay was used to analyze cell viability and cell proliferation. Logarithmically growing cells were incubated for 24 h with 0.4, 2.0, 4.0, or 10.0 µg/cm² Cr2O3 particles; 26.4, 66.0, 132, 264, 660, or 1320 µM (not shown) CrCl3; or 1.32, 2.64, 6.6, 13.2, 26.4, or 66 µM K2Cr2O7. The incubation solution was removed after 24 h and the cells were washed twice with PBS. The CellTiter-Glo® Luminescent Cell Viability Assay was carried out according to the manufacturer’s protocol. Briefly, complete medium and the equal volume of CellTiter-Glo® reagent were added to the cavities. The 96-well plate was transferred on an orbital shaker for 2 min to induce cell lysis. The plate was incubated for 40 min in the dark at room temperature to stabilize the luminescent signal, which was then recorded with a microplate reader TECAN® Infinite M200 Pro (TECAN Group, Maennedorf, Switzerland). Data were analyzed with Excel 2010 (Microsoft, Redmond, CA, USA). The ATP content as a measure for cell viability was expressed as percentage normalized to non-treated control cells. To test if soluble Cr(III) or Cr(VI) interfered with the ATP assay, the relevant concentrations from above were added to a standard curve of ATP (0.5, 1.0, 2.5, 5.0 µM).
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