Cells were seeded in 96-well plates. On the next day, cells were treated with 100 µL Caspase-Glo® 3/7 Reagent (Promega, Madison, WI, USA) for 0.5–3 h at 25 °C. Then, caspase-3/7 activity was quantitated with a luminometer. Untreated cells were used as a negative control.
Caspase glo 3 7 reagent
Manufactured by Promega
Sourced in United States, United Kingdom
The Caspase-Glo 3/7 reagent is a luminescent assay that measures the activities of caspase-3 and caspase-7, two key enzymes involved in the apoptosis (programmed cell death) pathway. The reagent contains a proluminescent caspase-3/7 substrate, which is cleaved by the active enzymes, resulting in the generation of a luminescent signal. This signal is proportional to the amount of caspase-3/7 activity present in the sample.
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Lab products found in correlation
Caspase glo 3 7 assay, by Promega (10 mentions)
Celltiter glo, by Promega (5 mentions)
Staurosporine, by Merck Group (4 mentions)
Celltiter glo reagent, by Promega (4 mentions)
Spectramax m5, by Molecular Devices (3 mentions)
Fluostar omega plate reader, by BMG Labtech (3 mentions)
Infinite 200 pro, by Tecan (3 mentions)
Annexin 5 fitc, by Biotium (2 mentions)
Attune nxt 4 laser cytometer, by Thermo Fisher Scientific (2 mentions)
D300e digital dispenser, by Tecan (2 mentions)
F200 pro microplate reader, by Tecan (2 mentions)
Infinite m200, by Tecan (2 mentions)
Caspase glo 3 7 assay system, by Promega (2 mentions)
Caspase glo 3 7, by Promega (2 mentions)
Caspase glo 3 7 kit, by Promega (2 mentions)
Infinite m1000 microplate reader, by GraphPad (2 mentions)
Cytochalasin d, by Promega (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (2 mentions)
Pherastar fs, by BMG Labtech (2 mentions)
181 protocols using caspase glo 3 7 reagent
1
Caspase-3/7 Activity Quantification
2
Measuring Neutrophil Apoptosis Induced by Terpenoids
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The cell apoptosis was analyzed by measuring the activity of proapoptotic caspases 3/7. Neutrophils (2 × 105 cells/well), suspended in solutions of FOH (trans,trans−3,7,11-Trimethyl−2,6,10-dodecatrien−1-ol; Sigma-Aldrich, St. Louis, MO, USA), FA (Echelon Biosciences Inc, Salt Lake City, UT, USA) or TR (Sigma-Aldrich, St. Louis, MO, USA) at variable concentrations, were placed in the wells of a 96-well white microplate and incubated for 1 h at 37 °C, at 5% CO2. Then, the cells were washed with PBS, and 100 µL of Caspase-Glo® 3/7 Reagent (Caspase-Glo® 3/7 Assay, Promega, Madison, WI, USA) was added to each well, the plate was gently mixed by shaking at 300 rpm for 30 s and the chemiluminescence was measured continuously for 2 h at 37 °C.
3
Evaluating Apoptosis in PDAC Cell Lines
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To evaluate the apoptotic rate for PDAC cell lines, 5000 cells/well were seeded in white 96-well plates and cultured for 24 h with Pladienolide-B or vehicle, and apoptotic rates were measured using Caspase-Glo 3/7 Reagent (Promega), following the manufacturer’s instructions [36 (link)]. For Annexin-V staining, floating and attached cells were pooled and resuspended in 1X Annexin-V staining buffer containing Annexin-V-FITC diluted 1:20 (Cat no. 29001, Biotium, Freemont, CA) and then, incubated for 20 min at room temperature prior to flow cytometric analysis. Cytometry data was acquired with an Invitrogen™ Attune™ NxT 4-laser cytometer with software version 3.1.1.
4
Caspase 3/7 Activation Assay
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Cells (5 × 103 cells/well) were seeded in a 96-well white plate and treated with MASM7, MFI8. Caspase 3/7 activation was measured after 6 h by addition of the Caspase-Glo 3/7 reagent according to the manufacturer’s protocol (Promega). Luminescence was detected by a F200 PRO microplate reader (TECAN). Caspase assays were performed in at least triplicate and the data normalized to vehicle-treated control wells. Dilutions of MASM7 or MFI8 were performed using a TECAN D300e Digital Dispenser from 10 mM stocks.
5
Caspase-Mediated Apoptosis Induction Assay
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Example 1
Caspase Activity assay: This is a cell assay to measure the induction of apoptosis in MOLP-8 (multiple myeloma), KMS-12-BM (multiple myeloma), MV4; 11 (acute myeloid leukemia), and NCI-H23 (non-small cell lung cancer) cells after 6 h treatment with Mcl-1 inhibitors. On the first day, 3000 (MOLP-8, KMS-12-BM, MV4; 11) or 1250 (NCI-H23) cells/well were seeded with 50 μL of growth media (IMDM+10% FBS+2 mM L-Glu for MV4; 11 and RPMI-1640+10% FBS+2 mM L-Glu for all others) in 384-well white microplates, and incubated overnight (37° C., 5% C02, 80% RH). On the second day, the cells were treated with Compound I using an ECHO acoustic liquid handler (10 point half-log serial dilution, 31.5 μM top concentration, 0.3% final DMSO concentration). After 6 h incubation (37° C., 5% C02, 80% RH), 25 μL of Caspase-Glo 3/7 reagent (Promega) was added into each well, and plates were incubated at room temperature for 30 min protected from light. Luminescence was recorded using an Infinite M200 microplate reader (Tecan) with a 100 ms integration time. EC50 values were calculated using GeneData analysis software and are shown in Table 2, below.
6
Spheroid Apoptosis Assay for PANC1 Cells
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PANC1 cells were seeded 5000 cells/ml in SCM media in Ultra-Low attachment T75 flask (Corning # 3814), and allowed to grow 5 days to form spheres. The spheres were treated with compound for 20 h. Spheres were pelleted by centrifugation and cell apoptosis was measured by addition of 100 μl of Caspase-Glo 3/7 reagent (Promega) directly to the pellet, incubation at room temperature for 30 min, and then transfer of 30 μl per well to each of three wells in a 384-well plate, to measure the luminescence signal with a ViewLux reader.
7
Quantitative Analysis of Caspase Activation
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For quantitative analysis of caspase activation, Caspase-Glo 3/7 Reagent (Promega) was added directly to Hepa1-6 cells in a 96- or 384-well plate at a volume equal to the sample volume. Luminescence was recorded after 30 min using a TECAN Infinite M200 plate reader. Background readings determined from wells containing culture medium only were subtracted. Relative caspase activity was calculated as 100% × luminescence reading of a treated sample/luminescence reading of a mock-treated control.
8
Evaluating Apoptosis Induction by NVS-ZP7-4
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To assess if NVS-ZP7-4 was stimulating the activity of caspase 3/7, an indicator of apoptosis, the Caspase-Glo 3/7 assay system (Promega, Fitchburg, WI) was used. Included in this assay were control compounds including Thapsigargin (Sigma), which is known to induce apoptosis, along with negative controls that included DMSO and NVS-ZP7-6 (inactive version of NVS-ZP7-4). The ARPE-19 was cultured, serum starved in 1% FBS overnight, then treated with IL-1 β (5ng/ml) alone or IL-1 β plus NVS-ZP7-4 (2 μM or 10 μM) for 24hr. Caspase-Glo 3/7 reagent (Promega) was prepared according to the manufacture’s protocol and added to the cells 24 h after compound addition. Each sample was incubated with the Caspase-Glo 3/7 reagent for 30 min and then luminescence (relative luminescence units) was read on the EnVision Multimode Plate Reader (PerkinElmer).
9
Caspase-3/7 Activity Assay in Lung Cancer Cells
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A total of 3–5×103 transfected HCC827 or Calu-3 cells were seeded in 96-well plates. After 48 h, cells were treated with Caspase-Glo® 3/7 reagent (Promega Corporation) according to the manufacturer's protocol; following agitation for 30 sec, cells were incubated for 2 h at room temperature. Fluorescence activity was analyzed using a GloMax® 96 Microplate Luminometer system (Promega Corporation). Relative fluorescent activity was quantified by setting the blank control to 1.
10
Caspase-3/7 Inhibition Assay
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