The MTC-SK cells were treated with 10 or 25 μg/ml of the different TG fractions; 5, 10 and 20 μM UA; DMSO or 5 μM CPT in 24-well plates. Following the incubation periods, 50 μl of each cell suspension was transferred into a 96-well white plate (Nunc, Roskilde, Denmark). Caspase activity was quantified using Caspase-Glo® assays (Promega, Mannheim, Germany). The activities of caspase-2, -3/7, -6, -8 and -9 were measured in the MTC-SK cells in each group. The Caspase-Glo® assays were performed, according to the manufacturer's instructions. Briefly, Caspase Glo® substrate was mixed with Caspase Glo® buffer (50 μl/well) in a 96-well plate according to the manufacturer's instructions (Promega). The samples were agitated for 30 sec and measured following a 1 h incubation at room temperature in the dark. To avoid cross reactions, 50 nM Ac-DEVD-CHO inhibitor (Promega) was added to the samples prior to addition of the reagents in the caspase-2, -6 and -9 assays. The emitted light was measured using a Lucy2 Typ 18610 luminometer (cat. no. 186101036; Mediators PhL) and the values were analyzed. Ham's F12+FBS (10%) medium was used as a blank control. Double measurements were obtained in each assessment.
Caspase glo assay
Manufactured by Promega
Sourced in United States, United Kingdom, Germany
The Caspase-Glo assay is a luminescent-based kit designed to measure caspase activity in cell-based assays. It provides a convenient way to quantify the activation of caspase enzymes, which are key mediators of apoptosis or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate format, by Merck Group (4 mentions)
Celltiter glo assay, by Promega (3 mentions)
Veritas microplate luminometer, by Promega (3 mentions)
Celltitre glo luminescent viability kit, by Promega (2 mentions)
Infinite m200 microplate reader, by Tecan (2 mentions)
Rnase a, by Qiagen (2 mentions)
Cell proliferation colorimetric assay, by Roche (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Matrigel basement membrane matrix, by BD (2 mentions)
Luciferin conjugated polypeptide substrate, by Promega (2 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (2 mentions)
Annexin v 7aad, by BioLegend (2 mentions)
Cd29 9eg7, by BD (2 mentions)
Pakts473, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment plate, by Corning (2 mentions)
White viewplate 384 microplate, by PerkinElmer (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
87 protocols using caspase glo assay
1
Caspase Activity Assay in MTC-SK Cells
2
Cell Viability and Caspase Assay
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Cell viability and caspase activity was determined using the CellTitre-Glo luminescent viability kit and caspase Glo assays (Promega, Madison, USA) as described in the manufacturer’s instructions.
3
Apoptosis Induction via Caspase Activation
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The induction of programmed cell death was determined by monitoring the activities of caspases-3/7, caspase-8, and caspase-9 by Caspase-Glo Assays (Promega, Madison, WI, USA) 24 h after treatment with 5, 10, and 20 μM of pancracine. Cells treated with 1 µM of doxorubicin were used as a positive control. The assay provides a proluminogenic substrate in an optimized buffer system. The addition of a Caspase-Glo Reagent results in cell lysis, followed by caspase cleavage of the substrate and the generation of a luminescent signal. A total of 1 × 104 cells were seeded per well using a 96-well plate format (Sigma-Aldrich, St. Louis, MO, USA). After treatment, the Caspase-Glo Assay Reagent was added to each well (50 μL/well) and incubated for 30 min before luminescence was measured using a Tecan Infinite M200 microplate reader (Tecan Group, Männedorf, Switzerland).
4
Caspase Activity Assay Protocol
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Activity of caspases-3/7 and -8 were assessed using the corresponding Caspase-Glo Assays in white-walled 96-well plates (Promega).
5
Ultrasensitive Caspase Activation Quantification
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For the quantification of caspase activation in control and Casp8−/− cells, an ultrasensitive and highly selective bioluminescence method based on Caspase-Glo® assays (Promega) was used. The activity of selected caspases (Caspase-Glo® 3/7 and Caspase-Glo® 6) was registered by a PMT head with a cooled photocathode (Hamamatsu 7421–40, Japan) working in photon-counting mode. The software for data acquisition and processing is an integral part of the Hamamatsu detector. The device was described in detail earlier (Ledvina et al., 2017 (link); Killinger et al., 2021 (link)). The bioluminescence reactions take place in eight 7 µl microvials tempered to 37°C. 10 µl of diluted cell suspension (1 × 105) were transferred into 30 µl of Caspase-Glo® lysis substrate and incubated for 20 min at 37°C for signal stabilization. Then lysate was added into 7-µl microvials and the bioluminescence signal was measured. The bioluminescence photon emission was counted in an interval of 10 s. The signal of 7 µl of Caspase-Glo® lysis substrate was taken as a blank.
6
Cell Viability and Apoptosis Assay
7
Anoikis and Caspase-Glo Assays
8
Determining Synergistic Drug Combinations
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Cell viability and caspase activity was determined using the CellTitre-Glo® luminescent viability kit and caspase Glo® assays (Promega, Madison, USA) as described in the manufacturer's instructions. Calcusyn Software Version 2.1 (Cambridge Computing Imaging Ltd, Cambridge, UK) was used to calculate the combination index (CI) using the percentage of cells affected. The Chou-Talalay method [62 (link)] is employed by Calcusyn for calculating the CI. The software uses a method based on the median effect equation (MEE) derived from the mass action law and correlates dose and effect to produce the CI value; anything less than one showing a level of synergy (closer the number to 0 shows a greater degree of synergy) or a number greater than 1 showing antagonism between the agents. In this case we used the percentage of non-viable cells after treatment (as determined by the CellTitre-Glo® assay described above) as the fractional affect. This data is input into the software along with the dose of all agents used in the combination treatment.
9
Assaying Caspase Activities in SW620 Cells
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The activities of initiator caspases-8 and -9 and effector caspases-3/7 were assayed using Caspase-Glo Assays (Promega, Madison, USA). The SW620 cells (seeded in a 96-well plate at a density of 30,000 cells/well) were cultivated in 12-well plates. After 4, 8 and 24 h incubation of cells in the medium containing the tested compounds pre-dissolved in DMSO at a concentration equal to their IC50 concentration, cells were lysed using a buffer containing 50 mM HEPES, 5 mM CHAPS and 5 mM DTT. The untreated control cells were incubated in medium containing only 0.1% DMSO. The lysates were collected into microtubes. Caspase-8, Caspase-9 and Caspases-3/7 reagents were prepared according to the manufacturer’s instructions. The lysates (25 μL in each well) were transferred into a white-walled 384-well-plate for the luminometer, at which point 25 μL of Caspase-8 or Caspase-9 or Caspases-3/7 reagents were added to each well. The plate was gently mixed for 30 seconds and then incubated for 30 minutes at room temperature. The luminescence was measured using the luminometer Infinite M200 (Tecan, Männedorf, Switzerland).
10
Cell Viability, Apoptosis, and Caspase Assay
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