Cells were plated in a 384-well white-walled, clear-bottom plate at 2500 cells per well in 100 µl and subjected to 4.5 µM doxycycline. Cells were incubated overnight to adhere to the plate, and were then treated at indicated concentrations. After cells were treated and the plate was incubated for the indicated time, Presto-blue was added at a volume of 1/10 total volume per well and cells were incubated for 10 minutes. The plate was then read for fluorescence at 560excitation/590emission. Caspase-Glo (Promega G8093) reagent was added and the plate was incubated for 30 minutes. The plate was then read for luminescence at 1000 milliseconds. Values were calculated as ([luminescence × 100]/fluorescence)/DMSOaverage.
Caspase glo
Manufactured by Promega
Sourced in United States, Germany, United Kingdom
Caspase-Glo is a luminescent assay system that measures caspase activity in cell-based apoptosis assays. The assay provides a homogeneous, plate-ready format that is designed to be sensitive, robust, and easy to use.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo, by Promega (17 mentions)
Lb960, by Berthold Technologies (3 mentions)
Celltiter blue, by Promega (2 mentions)
Lmaxii384, by Molecular Devices (2 mentions)
Cytation 3, by Agilent Technologies (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Apopladder ex, by Takara Bio (2 mentions)
Glomax multi detection system, by Promega (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Pluronic f 68 non ionic surfactant, by Thermo Fisher Scientific (1 mentions)
Genion luminometer, by Tecan (1 mentions)
Infinite f500 plate reader, by Tecan (1 mentions)
Flatbottom ultra low attachment plate, by Corning (1 mentions)
Round bottom ultra low attachment plate, by Corning (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Prism 5, by GraphPad (1 mentions)
Victor x3 plate reader, by PerkinElmer (1 mentions)
96 well flat bottomed plates, by PerkinElmer (1 mentions)
Biotek synergy 2 luminometer, by Agilent Technologies (1 mentions)
67 protocols using caspase glo
1
Cell Viability and Apoptosis Assay
2
Exendin-4 Effects on Cardiomyocyte Remodeling
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To investigate direct effects of exendin-4 on cardiomyocyte remodelling, a series of studies were conducted in rat ventricular H9c2 cardiomyoblasts and mouse atrial HL-1 cardiomyocytes, to assess actions on cell hypertrophy and apoptosis, respectively. H9c2 cardiomyoblasts were maintained in DMEM containing 10 % FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. At passage, they were plated, cultured to ~50 % confluency and serum-starved for 24 h prior to incubation with phenylephrine (1 μmol/L for 96 h) to induce hypertrophy in the presence or absence of exendin-4 (0.1 μmol/L) [33 (link)]. H9c2 cardiomyoblast cross-sectional area was quantified by blinded digital image analysis (NIS-Elements) as an index of cell hypertrophy. HL-1 cells were maintained in Claycomb media containing 10 % FCS, 0.1 mmol/L norepinephrine, 2 mmol/L l -glutamate, 100 U/ml penicillin and 100 µg/ml streptomycin. At passage, they were plated and cultured to sub-confluency (70–80 %) prior to incubation with doxorubicin (5 μmol/L for 24 h) to induce apoptosis in the presence or absence of exendin-4 (0.1 μmol/L) [34 (link)]. Caspase 3/7 activity (Caspase-Glo®, Promega UK) was quantified as a measure of apoptosis and data normalised to cell viability assessed by CellTiter-Blue® assay (Promega, UK).
3
Targeted ADC Disrupts Bcl-xL Complexes
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Example 7
A431 cells were plated at 50,000 cells per well in 96 well plates (Costar, 3610) in growth media. After 24 hours in culture at 37° C., ADCs were added to wells and incubated at 37° C. in a CO2 incubator for 24 hours. After incubation, 100 μL of Caspase-Glo® (caspase luminescent assay) 3/7 Assay reagent (Promega, G8093) was added to each well and shaken for 10 minutes. Plates were then incubated at 37° C. for 20 minutes. Caspase 3/7 activity was assessed using a Victor luminescence plate reader (Perkin Elmer).
While a non-targeting ADC (MSL109 hIgG-AAA) or AM2 failed to disrupt Bcl-xL-BIM complexes, AM2-AAA treatment resulted in efficient complex disruption in both cell lines. These results indicate that the AAA warhead was specifically delivered via AM2-AAA to EGFR expressing cells (See
The ability of AM2-AAA to promote caspase activation, a downstream consequence of Bcl-xL inhibition, was also assessed in the A-431 cells. AM2-AAA, but not AM2 or the non-targeting MSL109 hIgG-AAA induced caspase activation supporting EGFR-dependent on mechanism activity of the targeted ADC.
4
Caspase Activity Measurement Protocol
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For measuring Caspase activity, the Caspase-Glo® assay from Promega was used according to the manufacturer's recommendations.
5
Caspase Activity Measurement by Luminescent Assay
6
Measure Cell Viability and Caspase Activity
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Viability of the cells was measured using ATP-based Cell Titer Glo assays (Promega) according to the manufacturer’s instructions using a plate reader (BioTek’s Synergy H1, Winooski, VT, USA). All experiments were performed in triplicates. To further validate KDM2B phenotype, stable cell lines of shKDM2B or shControl cells were expanded for three to four passages. Accordingly, 10 000 cells per well were seeded in triplicates and treated with TRAIL (0–100 ng/ml) for 24 h. To test caspase inhibition, each caspase inhibitor was used at 20 μM final concentration; cells were cotreated with inhibitors and TRAIL. For caspase activity measurements, cells were seeded in 96-well plates as 10 000 cells per well in triplicates, treated with TRAIL (0–100 ng/ml) for 3 h and caspase-8 and -3/7 activities were measured using Caspase Glo (Promega) assays according to the manufacturer’s instructions.
7
Caspase-Glo Assay for Compound Cytotoxicity
8
Caspase Activity Assay in A549 Cells
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The effects of Bi-L-RhamBet, Hed, and STS on caspase (3/7, 8, and 9) activity was determined using Caspase-Glo (Promega). Treatments were also performed with Bi-L-RhamBet associated with 10 μM of antimycin A, 10 mM of malonate and 10 μM rotenone. Controls did not have added antimycin, malonate, or rotenone. Briefly, 96-wells plates were seeded with 2×104 A549 cells per well and incubated for 14 h. After treatments with different compounds at periods of 2, 4, 6, 8, and 10 h, caspase substrate was added and plates were incubated in the dark for 1 h. Luminescence was measured by Cytation3.
9
Apoptosis Assay in Min6 Cells
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A Caspase-Glo 3/7 (Caspase-Glo®, Promega) apoptosis detection kit was used to analyse Min6 cell apoptosis as described previously [7 (link)]. Groups of 2 × 104 Min6 cells were maintained in culture in DMEM containing 2% FBS in the absence or presence of exogenous Wnt5a (0.05 μg/ml) for 48 h and were subsequently incubated for 20 h in the absence or presence of a cytokine cocktail (1 U/μl TNF-α, 0.05 U/μl IL-1β, and 1 U/μl IFN-γ) to induce apoptosis. Apoptosis was assessed by measuring caspase-3/7 activity as described previously [7 (link)].
10
Caspase-Glo Apoptosis Assay
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Cells were plated and cultured as for SRB assay. After 24 – 48 hours of incubation, Caspase Glo ® (Promega, Madison, Wisconsin, USA) was added to the media and incubated according to manufacturer's protocol. Luminescence was measured with a Genion luminometer (Tecan, Crailsheim, Germany). All measurements were carried out in triplicate/quadruplicate wells for at least two times and a representative example is shown.
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