We seeded 2.5 × 103 DCISCAF2cy expressing different shRNAs in non-FBS in DMEM/F12 on a low attachment 96-well plate. Apoptotic cell death was visualized and measured 6 h after seeding the cells using Caspase-3/7 Green Apoptosis Assay Reagent (Essen BioScience) and IncuCyte ZOOM (Essen BioScience). Caspase-3/7 activation was measured by counting the number of positive caspase-3/7 (green) objects and cell confluence (phase), as shown in Fig 4G .
Caspase 3 7 green apoptosis assay reagent
Manufactured by Sartorius
Sourced in Germany
The Caspase-3/7 Green Apoptosis Assay Reagent is a lab equipment product designed to detect and measure apoptosis, a form of programmed cell death. The reagent contains a fluorogenic substrate that becomes brightly fluorescent upon cleavage by caspase-3 and caspase-7 enzymes, which are activated during apoptosis. This allows for the quantification of apoptotic cells in a sample.
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Lab products found in correlation
Incucyte zoom, by Sartorius (4 mentions)
Cytotox green reagent, by Sartorius (2 mentions)
Celltrace far red proliferation kit, by Thermo Fisher Scientific (2 mentions)
Incucyte s3 machine, by Sartorius (2 mentions)
Caspase 3 7 green apoptosis assay reagent, by BD (1 mentions)
Anti fas antibody, by BD (1 mentions)
Incucyte zoom system, by Sartorius (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Cellbind, by Corning (1 mentions)
Incucyte s3 live imaging system, by Sartorius (1 mentions)
Apotox glo, by Promega (1 mentions)
Glomax discover system, by Promega (1 mentions)
14 protocols using caspase 3 7 green apoptosis assay reagent
1
Caspase-3/7 Apoptosis Assay for DCIS Cells
2
Cell proliferation and apoptosis assay
3
Real-Time Cell Cytotoxicity Assay
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For cytotoxicity death assay, cells were seeded in 96-well plates in low density (5,000 cells/well) and incubated overnight. 1000X Cytotox Green Reagent or Caspase 3/7 Green Apoptosis Assay Reagent (Essen Bioscience) was diluted in medium and working dilutions of the drug were prepared in Cytotox Green or Caspase3/7 Green supplemented media. After treatment, plates were loaded in IncuCyte Zoom and images were acquired in real-time for phase to quantify growth. Activity of green reagent was simultaneously acquired at the green channel to quantify death. IncuCyte Zoom software was used for the analysis and data export.
4
Caspase 3/7 Activation in Lung Myofibroblasts
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Kinetic estimation of caspase 3/7 activity was performed using the real-time imaging system IncuCyte ZOOM (Essen BioScience, Ann Arbor, MI, United States). Activation of caspase-3/7 in cells undergoing apoptotic death cleaves the caspase-3/7 substrate to produce nuclear green-fluorescence (Caspase-3/7 Green Apoptosis Assay Reagent [Essen Bioscience]). Primary lung-resident myofibroblasts or fibrocytes were prepared from normal or fibrotic lung tissue and cultured in a 12-well plate to 50–60% confluency. After growing overnight in low serum-containing MEM media, they had adapted to low-serum conditions. They were then treated with media containing either Caspase 3/7 Green Apoptosis Assay Reagent at a final concentration of 5 μM/mL or Caspase 3/7 Green Apoptosis Assay Reagent and anti-Fas antibody (BD Biosciences) at a final concentration of 250 ng/mL. Time-lapse fluorescence imaging was performed using the IncuCyte ZOOM system (Essen BioScience); 9 images per well at 20× magnification were collected every 2 h for 24–48 h. The average number of green objects produced by the apoptotic cells were measured using IncuCyte ZOOM software 2015A.
5
Assessing Long-term Cell Viability and Proliferation
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For long-term colony-formation assay, the cells were seeded with densities between 10 and 20,000 cells per well, depending on the cell line. Cells were treated with the indicated doses of the drugs which were refreshed every 2–3 d. At the end of the assay, cells were fixed with 2% of formaldehyde (Millipore) in PBS, stained with 0.1% crystal violet (Sigma-Aldrich) in water and scanned. For proliferation assays cells were plated in 96 or 384-well plates with densities between 125 and 1,000 cells per well. The cells were treated the following day using a HP D300 Digital Dispenser and drugs and medium were refreshed every 2–3 d. Plates were incubated at 37°C and images were taken every 4 h using the IncuCyte live cell imaging system. Confluency was calculated to generate growth curves. For apoptosis assay, caspase-3/7 green apoptosis assay reagent (#4440, 1:1,000; Essen Bioscience) was added to each well. Percentage of apoptotic cells was calculated by dividing the caspase-3/7 green signal by the confluence.
6
High-throughput Live-cell Apoptosis Assay
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Cell lines were seeded into 384-well plates at a density of 500 to 2,000 cells per well, depending on growth rate and the design of the experiment. Drugs were added at the indicated concentrations using the HP D300 Digital Dispenser (HP). Cells were imaged every 4 hours using the Incucyte ZOOM (Essen Bioscience). Phase-contrast images were analyzed to detect cell proliferation based on cell confluence. For the cell apoptosis assay, caspase-3/7 green apoptosis assay reagent (Essen Bioscience, #4440) was added to the culture medium and cell apoptosis was analyzed based on green fluorescent staining of apoptotic cells. The percentage of apoptotic cells was quantified based on images generated from biological triplicates.
7
Quantifying Caspase-3/7 Rescue in FSHD Myotubes
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Example 6
2° Assay: Live Cell Imaging of Caspase 3/7 Cleavage Dye Fluorescence Through Myotube Formation: GBC0905 (Rebastinib) rescues FSHD patient-derived myotubes from DUX4-induced death by Caspase 3/7-mediated apoptosis.
Methods:
17MB026 primary FSHD-affected patient myoblasts (obtained from the University of Rochester FSHD Biorepository) were thawed and cultured as described in Rickard et al., 2015 in primary myoblast medium. Cells (4000 per well) were seeded to a collagen-I coated 96-well plate in 100 μL primary myoblast medium. Medium was changed every other day until cells reached 80% confluence followed by a switch to 100 μL per well primary myotube medium containing 1:5000 diluted Caspase 3/7 Green Apoptosis Assay Reagent (Essen Bioscience). GBC0905 (Rebastinib, 10 nM-3 μM range of concentrations) of or DMSO vehicle was delivered to each well. Cells were imaged in the phase and green channels every six hours for four days to quantify green intensity×area per image. Triplicate wells of each condition were averaged.
Results:
As shown in
8
Co-culturing HSPCs with MSCs
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FACS-purified human CD34+ HSPCs were sorted directly into cytokine-free sterile RPMI medium supplemented with 10% FBS, 1% penicillin–streptomycin and 0.1% amphotericin B and plated in 48-well plates previously seeded with low-passage (P ≤4) healthy BM-derived human MSCs. Co-cultures were incubated at 37 °C in a 5% CO2 atmosphere. After treatment, cells were harvested and stained for quantitative flow cytometric analysis using the antigen panel detailed in Supplementary Table 1 , with AccuCheck Counting Beads (Thermo Fisher Scientific) added to each tube.
In live microscopy assays, FACS-purified Lin−CD34+ cells were sorted into PBS and stained with 0.5 µM IncuCyte CytoLight Rapid Red Reagent (Essen Biosciences). The cells were then washed and plated in 96-well plates pre-seeded with MSCs. After 12–16 h in culture, the indicated drugs were added to each well in the presence of Caspase-3/7 Green Apoptosis Assay Reagent (1:1,000 dilution; Essen Biosciences). Co-cultures were incubated in an IncuCyte S3 Live Cell Analysis System (Essen Biosciences) placed in an incubator at 37 °C and 5% CO2. Live microscopy images of the co-cultures were captured every hour using the IncuCyte S3 software (v2017A, Essen Biosciences) at ×20 magnification.
In live microscopy assays, FACS-purified Lin−CD34+ cells were sorted into PBS and stained with 0.5 µM IncuCyte CytoLight Rapid Red Reagent (Essen Biosciences). The cells were then washed and plated in 96-well plates pre-seeded with MSCs. After 12–16 h in culture, the indicated drugs were added to each well in the presence of Caspase-3/7 Green Apoptosis Assay Reagent (1:1,000 dilution; Essen Biosciences). Co-cultures were incubated in an IncuCyte S3 Live Cell Analysis System (Essen Biosciences) placed in an incubator at 37 °C and 5% CO2. Live microscopy images of the co-cultures were captured every hour using the IncuCyte S3 software (v2017A, Essen Biosciences) at ×20 magnification.
9
Apoptosis Monitoring in Leukemia Cells
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KMM1 or LP1 cells were plated into 96-well plates and treated with S63845 (50 nM) and/or venetoclax (100 nM) in the presence of Caspase-3/7 Green Apoptosis Assay Reagent (Essen BioScience) according to the manufacturer’s instructions. Culture plates were placed into the IncuCyte S3 live-cell analysis system and three fields per well were scanned every 30 min during 15 h. Caspase -3/7 green reagent fluorescence was normalized to cell confluence.
10
Real-time Cytotoxicity Monitoring Assay
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For cytotoxicity death assay, cells were seeded in 96-well plates in low density (5,000 cells/well) and incubated overnight. 1000X Cytotox Green Reagent or Caspase 3/7 Green Apoptosis Assay Reagent (Essen Bioscience) was diluted in medium and working dilutions of the drug were prepared in Cytotox Green or Caspase3/7 Green supplemented media. After treatment, plates were loaded in IncuCyte Zoom and images were acquired in real-time for phase to quantify growth. Activity of green reagent was simultaneously acquired at the green channel to quantify death. IncuCyte Zoom software was used for the analysis and data export
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