The 15 × 103 cells were plated in triplicate/quadruplicate in 96-well, white, flat-bottom, cell culture-treated assay plates in three independent experiments. After 24 h, normal growth media was replaced with 100 μL of media supplemented with A1mcMMAF (10–100 μg/mL). Forty-eight hours after the first drug exposure, 20 μL of a viability/cytotoxicity substrate containing 10 μL of GF-AFC and 10 μL of bis-AAF-R110 were added to each well (ApoTox-Glo, Triplex Assay; Promega) to enable simultaneous measurement of each parameter with minimal degradation of proteases secreted at early time points. After 30 min at 37 °C, the fluorescence was measured using a Fluostar Optima plate reader (BMG Labtech, Ortenberg, Germany). To measure apoptosis, 100 μL of Caspase-Glo 3/7 reagent was added to each well and incubated at room temperature for 30 min (ApoTox-Glo, Triplex Assay; Promega). Luminescence was then measured using the Fluostar Optima plate reader.
Apotox glo triplex assay
Manufactured by Promega
Sourced in United States, United Kingdom, Germany, Switzerland
The ApoTox-Glo Triplex Assay is a multiplexed luminescent assay that simultaneously measures cell viability, cytotoxicity, and apoptosis within the same sample. This assay provides a versatile tool for evaluating cellular responses to various treatments or stimuli.
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Lab products found in correlation
Glomax multi detection system, by Promega (5 mentions)
Staurosporine, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Infinite m200, by Tecan (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Varioskan flash, by Thermo Fisher Scientific (2 mentions)
Mitochondrial toxglo assay, by Promega (2 mentions)
Clariostar microplate reader, by BMG Labtech (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Victor3 multilabel plate reader, by PerkinElmer (1 mentions)
Clear bottom 96 well plate, by Corning (1 mentions)
Modulus microplate reader, by Promega (1 mentions)
Celltiter glo luminescent assay, by Promega (1 mentions)
Z vdvad fmk, by R&D Systems (1 mentions)
Brdu assay, by Cell Signaling Technology (1 mentions)
Prism 6, by GraphPad (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Black 96 well plate, by Corning (1 mentions)
113 protocols using apotox glo triplex assay
1
Cytotoxicity and Apoptosis Measurement
2
ApoTox-Glo Triplex Assay for Apoptosis
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The ApoTox-Glo triplex assay (Promega, G6320) is more complex and is centered on the proteolytic action of biomarkers present in the cell. It was used to evaluate cell viability, cell cytotoxicity, and caspase 3/7 activation (a marker of apoptosis) 24, 48 or 72 h post-irradiation. Briefly, 100 µL of cell suspension (containing 20, 000 cells per 100 µL) was introduced into an opaque-walled 96-well plate and 20 µL of mixed glycyl phenylalanyl-amino fluorocoumarin (GF-AFC) and bis-alanylalanyl-phenylalnyl-rhodamine 100 (bis-AAFR110) substrate (viability/cytotoxicity reagent) was added. This mixture was combined for 30 s by orbital shaking (350 rpm) followed by incubation at 37 °C for 1 h, then fluorescent signals were measured using the PerkinElmer Victor3™ multilabel plate reader at 400EX/505EM (filters for viability) and 485EX/520EM (filters for cytotoxicity). Caspase 3/7 activity was subsequently quantified by adding 100 µL Caspase-Glo® 3/7 reagent to all wells, followed by incubation for 30 min at room temperature and measurement of luminescence in RLU. Background fluorescence was eliminated by subtracting the fluorescence of media without cells.
3
Multiplex Assay for Cell Viability, Cytotoxicity, and Apoptosis
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The ApoTox-Glo triplex assay (Promega, Promega BioSciences, LLC, San Luis Obispo, CA, USA) was performed according to our previously defined procedure22 (link). Briefly, the BV2 microglial cell lines were seeded in 96-well plates (1 × 105 cells/well) in 200 μl of DMEM media. Cells received respective drug treatments, and viability/cytotoxicity reagents containing a GF-AFC substrate and bis-AAF-R110 substrate were added to all wells. Cells were agitated for 30 sec and incubated for 1 hr at 37 °C. Fluorescence spectra were measured at 400/505 nm for viability assays and 485/520 nm for cytotoxicity assays. Caspase3/7 activity was assessed by the addition of caspase-Glo 3/7 reagent (100 μl) to all wells, and luminescence was measured at 485/520 nm to determine caspase activation. The results were calculated as % cell viability, % cytotoxicity, and % caspase-3/7 activity.
4
Cytotoxicity and Apoptosis Measurement
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Viability, cytotoxicity and apoptosis were measured using the ApoTox‐Glo Triplex Assay (Promega, UK) according to manufacturer's instructions. Cells were cultured at 20,000 cells/well in clear‐bottom 96‐well plates (Costar, Corning Life Sciences, NY, USA). Fluorescence and luminescence readings were made using a Tecan plate reader, and data were normalised to the medium‐only sample. To normalise well‐to‐well variability, cytotoxicity and caspase measurements were represented as normalised to viability measurements (dead/live cell ratio and apoptotic/live cell ratio). Cells subjected to 3 h of 1 μM staurosporine treatment were used as positive control.
5
CD40 and Cytokine-Induced B-Cell Activation
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Cells were cultured seeded in 1 µg/ml anti-human CD40 and 10 ng/ml IL-4 and IL-21 for 48 h. Medium was removed and 1.5 × 105 cells were seeded into 96-well plates in replicates with anti-CD40 and IL-4/IL-21 or controls. ApoTox-Glo Triplex Assay (Promega, Walldorf, Germany) was applied for analysis according to the manufacturer’s recommendation.
6
Telomerase-Immortalized Fibroblasts Reveal Myogenic Potential
7
Cell Viability and Apoptosis Assays
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BSG neurospheres were generated, cultured, and treated as above for cell proliferation. For cell viability, the CellTiter-Glo Luminescent Assay (Promega) was performed, and for apoptosis, the Promega ApoTox-Glo Triplex Assay (# G6321) was used, both according to the manufacturer’s instructions. Luminescence was read using a Turner Biosystems Modulus Microplate Reader. Data analysis and statistics were performed as above for cell proliferation.
8
Cytotoxicity of GANT58 in MDA-MB-231 Bone Cells
9
Caspase-2 Inhibition Modulates Adipocyte Function
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Mouse adipose tissue stem cells were isolated from caspase-2-deficient and WT mice as previously described.60 3T3-L1 cells were differentiated into adipocytes,61 (link) and differentiation confirmed with Oil red staining. Differentiated adipocytes were treated with 1 mM palmitate without or with 20 μM caspase-2 inhibitor Z-VDVAD-FMK (R&D Systems, Minneapolis, MN, USA) for 48 h.
Apoptosis was measured using ApoTox-Glo Triplex Assay (Promega, San Luis Obispo, CA, USA) and proliferation using BrdU assay (Cell Signaling, Danvers, MA, USA).
Apoptosis was measured using ApoTox-Glo Triplex Assay (Promega, San Luis Obispo, CA, USA) and proliferation using BrdU assay (Cell Signaling, Danvers, MA, USA).
10
Evaluating ADI-PEG 20 Cytotoxicity in Cancer Cells
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Cancer cell lines were cultured according to manufacturer's instructions. Cells were treated with increasing concentrations of ADI-PEG 20 and analyzed after 24 h, 48 h or 72 h incubation.
The effect of ADI-PEG 20 on cell viability and caspase 3/7 induction was analyzed by ApoTox-Glo triplex assay (Promega).
The effect of ADI-PEG 20 on cell viability and caspase 3/7 induction was analyzed by ApoTox-Glo triplex assay (Promega).
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