Since the previous study [18 (link)] established that the extracts at 100 μg/mL did not induce significant cytotoxicity on human embryonic kidney cells and macrophage, this indicated that the extracts may not have any cytotoxic effect to non-cancerous cells. Hence, concentrations used were up to 100 μg/mL. Cells were plated in 384-well plates for 24 h followed by LP, LN, SP and SN treatment at 1-100 μg/mL for 72 h incubation as per established in previous studies [11 (link)]. As a comparison, we also tested vitexin and isovitexin (Sigma Aldrich, USA), the pure compounds that were commonly isolated Clinacanthus nutans extracts [13 (link)]. CellTitre-Glo® Luminescent Cell Viability Assay kit (Promega, USA) was used to quantify the cell viability based on the luminescence readings recorded using SpectraMax M3 Multi-Mode Microplate Reader (Radnor, USA). IC50 values were calculated based on the dose-response curve generated. The experiments were validated using with 100 μg/mL gemcitabine and 5-fluorouracil, the first line clinically used anti-cancer agents.
Celltitre glo luminescent cell viability assay kit
Manufactured by Promega
Sourced in United States
The CellTiter-Glo® Luminescent Cell Viability Assay kit is a quantitative method for determining the number of viable cells in a culture based on the measurement of ATP, an indicator of metabolically active cells. The assay involves the addition of a single reagent that induces cell lysis and generates a luminescent signal proportional to the amount of ATP present.
Automatically generated - may contain errors
Lab products found in correlation
Isovitexin, by Merck Group (1 mentions)
Vitexin, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
Cytation 3 plate reader, by Agilent Technologies (1 mentions)
Senescence detection kit, by Abcam (1 mentions)
5 fluorouracil, by Merck Group (1 mentions)
5 protocols using celltitre glo luminescent cell viability assay kit
1
Cytotoxic Evaluation of Clinacanthus nutans Extracts
2
Ox-LDL Impacts on ATP Levels
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The cells were inoculated in a six-well plate. ox-LDL (12.5 μl and final concentration was 50 μg/ml) was added to the ox-LDL treatment group when the cells grew to 70% confluent. Then the same amount of solvent was added to the control group and culture for 48 h. ATP levels were determined using the Cell Titre-Glo Luminescent Cell Viability Assay Kit (Promega), as previously described (35 (link)). All tests were repeated three times.
3
Mitochondrial Oxidative Stress Evaluation
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Podocytes were incubated with HBSS buffer containing 3 μM MitoSox (Life Technologies) for 20 min at 37 °C in a non-CO2 incubator. MitoSox fluorescence was analyzed by flow cytometry with help from UT-MDACC FCCICF Core, as previously described (25 ). Cellular ATP levels were measured using the CellTitre-Glo Luminescent Cell Viability Assay Kit (Promega). ATP levels were normalized by DNA concentration in each well. DNA concentration was measured using CyQUANT Cell Proliferation Assay Kit (Molecular Probes). Oxygen consumption rate was measured using Seahorse XFe96 Analyzer (Agilent) as previously described (25 ) and subsequently normalized using the CyQUANT Cell Proliferation Assay Kit for DNA concentration.
4
Cell Proliferation and Senescence Assays
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Cellular proliferation after treatment was measured using a CellTitre-Glo Luminescent Cell Viability Assay kit (Promega) according to manufacturer’s instructions. Cells were seeded in 96-well plates at 5 × 103 cells per well overnight before drug treatment and luminescence measured on a Cytation 3 plate reader (BioTek). After combination treatment senescence was measured by β-galactosidase staining using an Abcam Senescence Detection Kit (Abcam). Cells were plated and fixed in 6-well plates after combination treatment and stained with X-gal. Positively stained cells were identified under a light microscope.
5
Cytotoxicity of Cud C Compound
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Concentration-response curves and IC50 values were calculated using the Cell Titre-Glo® Luminescent Cell Viability Assay kit (Promega, USA). Briefly, cells were plated in 96-well plates for 24 hours followed by Cud C treatment for 72 hours Luminescence was measured using SpectraMax M3 Multi-Mode Microplate Reader (Radnor, USA). The experiments were validated using CRC treated with 5-fluorouracil (Sigma-Aldrich, USA), the clinically used anti-cancer agent. Cell viability was expressed as a percentage of the vector-treated control.
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