Activation of caspase-3, caspase-8, and caspase-9 in living cells was evaluated by the CaspGLOW™ fluorescein active caspase staining kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocols. Briefly, treated cells were incubated with medium containing caspase fluorescent substrates for 2 hours at 37°C. After trypsinization and centrifugation, samples were resuspended in wash buffer and analyzed on a Canto II cytometer. The acquired data were then analyzed using the FCS express software.
Caspglow fluorescein active caspase staining kit
Manufactured by Abcam
Sourced in United States
The CaspGLOW fluorescein active caspase staining kits are a set of laboratory reagents used to detect and measure the activity of caspase enzymes in cells. These kits utilize a fluorescein-conjugated caspase inhibitor that binds to active caspases, allowing for the visualization and quantification of caspase activity through fluorescence-based techniques.
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Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (4 mentions)
Mitotracker green, by Thermo Fisher Scientific (4 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Accuri c5 cytometry, by BD (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
P ire1α, by Cell Signaling Technology (1 mentions)
Rhod 2, by Thermo Fisher Scientific (1 mentions)
Fluo 3, by Thermo Fisher Scientific (1 mentions)
Dioc6, by Thermo Fisher Scientific (1 mentions)
Bcl x, by Santa Cruz Biotechnology (1 mentions)
Gadd34, by Cell Signaling Technology (1 mentions)
P c jun, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Accuri c5 flow cytometer, by BD (1 mentions)
Annexin 5 fitc conjugate, by BioLegend (1 mentions)
11 protocols using caspglow fluorescein active caspase staining kit
1
Caspase Activity Assay in Cells
2
Caspase Activity Assay of ACE Polysaccharides
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Activities of caspase-3, caspase-8 and caspase-9 were determined with a CaspGLOW Fluorescein Active Caspase Staining Kit (BioVision, USA). In brief, cells were pre-incubated with ACE polysaccharides (25 μg/mL), ACE/CS (ACE polysaccharides = 13.2 μg/mL) and ACE/S (ACE polysaccharides = 21.2 μg/mL) for 48 h and counted to 1.0 × 106 cells/60-mm dish. After centrifuging at 400× g for 10 min, the cell pellets were lysed and kept on ice for 10 min. Supernatants were collected after centrifuging at 10,000× g at 4°C for 3 min and DEVD-pNA (Asp-Glu-Val-Asp p-nitroaniline, for caspase-3), IETD-pNA (Ile-Glu-Thr-Asp p-nitroaniline, for caspase-8), LEHD-pNA (Leu-Glu-His-Asp p-nitroaniline, for caspase-9) being added to a final concentration of 50 mM. Each sample was further incubated at 37°C for 1 h in a water bath and fluorescence was detected by a FACSCalibur Flow Cytometer with excitation at 485 nm and emission at 520 nm based on a minimum of 105 cells per sample.
3
Multiparametric Analysis of B Cell Proliferation and Apoptosis
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For cell proliferation analysis by cell tracking dye dilution, primary B cells were pulsed with 5 µM CellTrace Violet (Thermo Fisher Scientific) for 10 min at 37°C. Apoptosis analysis was performed by using CaspGLOW Fluorescein Active Caspase Staining Kit (BioVision) according to the manufacturer’s instructions. Samples were acquired on a LSRFortessa cell analyzer (BD Biosciences). Mitochondrial mass and membrane potential were measured via staining with MitoTracker Green and DeepRed (Thermo Fisher Scientific), respectively, according to the manufacturer’s instructions. Samples were acquired on a LSRFortessa cell analyzer (BD Biosciences).
4
Multiparametric Analysis of B Cell Proliferation and Apoptosis
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For cell proliferation analysis by cell tracking dye dilution, primary B cells were pulsed with 5 µM CellTrace Violet (Thermofisher) for 10 min at 37 °C. Apoptosis analysis was performed by using CaspGLOW™ Fluorescein Active Caspase Staining Kit (BioVision) according to the manufacturer's instructions. Samples were acquired on a LSRFortessa cell analyzer (BD-Biosciences). Mitochondrial mass and membrane potential were measured via staining with MitoTracker Green and DeepRed (ThermoFisher), respectively, according to the manufacturer's instructions. Samples were acquired on a LSRFortessa cell analyzer (BD-Biosciences).
5
Caspase Activity Measurement
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After treatment with MeCDDA for 24 h, cells were collected for measurement of caspase-3, 8, and 9 activities using the appropriate CaspGLOW™ Fluorescein Active Caspase Staining Kits (Biovision, Milpitas, CA, USA) according to the manufacturer’s specifications.
6
Caspase Activation by Agelasidine A
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The cells were seeded into six-well plates at a density of 2 × 105 cells/well and treated with (−)-agelasidine A (35, 70, and 140 μM) for 24 h. Caspase-3, -8, and -9 activities were measured using the appropriate CaspGLOW fluorescein active caspase staining kits (Biovision, Milpitas, CA, USA) according to the manufacturer’s protocol and measured by flow cytometry on an Accuri C5 cytometry (BD Biosciences, Ann Arbor, MI, USA).
7
Caspase-3 and -7 Activity Assay
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The cells were seeded at 2 × 105 cells/well in a 6-well plate overnight and were incubated in culture medium containing 11-dehydrosinulariolide at different concentrations (0, 10, 25, and 50 μM) for 24 h and were collected for the measurement of caspase-3 and -7 activities using the appropriate CaspGLOW™ Fluorescein Active Caspase Staining Kits (Biovision, Milpitas, CA, USA) according to the manufacturer’s specifications.
8
Endoplasmic Reticulum Stress Pathway
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Primary antibodies against Grp78, GADD34, CHOP, p-Ire-1 α, p-PERK-1 and p-c-Jun were purchased from Cell Signaling Technology (Danvers, MA); Bax, bcl-2, bcl-x, actin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CaspGLOW Fluorescein Active Caspase Staining Kits were purchased from Biovision (Milpitas, CA). Cell culture media were obtained from Hyclone (South Logan, UT). DiOC6, Fluo-3 and Rhod-2 were purchased from Invitrogen (New York, USA). Western blot chemiluminescence reagents were purchased from Millipore (Boston, MA). The lentiviral siRNAs, shGADD34 (TRCN03041) and shGFP (TRCN 072178) were purchased from National RNAi Core Facility (Taipei, Taiwan) and DNA sequence is shown in Supplemental Table 3 . All other chemicals were obtained from Bio-Rad (Richmond, CA), USB (Darmstadt, Germany) or Sigma Chemical (St. Louis, MO) and were the molecular biologic grade or higher. The use of the toxic chemicals followed the rules of Toxic Chemical Substances Control Act of Taiwan. All the following experiments also followed the guidelines of Good Laboratory Practice of Taiwan.
9
Apoptosis Induction and Caspase Activation in NPC Cells
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NPC cells were treated with DA-containing medium for at least 24 h before being harvested for staining with propidium iodide (PI) (50 µg/ml) and Annexin V-FITC (20 µg/ml) (Biolegend) at room temperature for 15 min. Apoptotic cells were quantified by an Accuri C5 flow cytometer (BD Biosciences).
CaspGLOW fluorescein active caspase staining kits (Biovision) were used to measure caspase activities, including caspase-3, -8, and -9. Cells were treated with DA-containing medium for 24 h, harvested, centrifuged at 1,200 rpm, washed with phosphate-buffered saline (PBS), and finally incubated with a caspase staining kit according to the manufacturer’s instructions before being analyzed by flow cytometry. In addition, Z-DEVD-FMK, an inhibitor of caspase-3, was used to treat the cells 2 h before DA treatment to rescue NPC cell viability, which was measured by MTT assays. For cell cycle analysis, intracellular PI staining was used to determine the DNA content linked to the cell cycle status.
CaspGLOW fluorescein active caspase staining kits (Biovision) were used to measure caspase activities, including caspase-3, -8, and -9. Cells were treated with DA-containing medium for 24 h, harvested, centrifuged at 1,200 rpm, washed with phosphate-buffered saline (PBS), and finally incubated with a caspase staining kit according to the manufacturer’s instructions before being analyzed by flow cytometry. In addition, Z-DEVD-FMK, an inhibitor of caspase-3, was used to treat the cells 2 h before DA treatment to rescue NPC cell viability, which was measured by MTT assays. For cell cycle analysis, intracellular PI staining was used to determine the DNA content linked to the cell cycle status.
10
Caspase Activity Assay in Cells
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Cells were seeded in 6-well plates (2×105 cells/well) and then treated with the indicated concentrations of kurarinone for 24 h. Cells were harvested and tested for caspase-3, −8, and −9 activities using the appropriate CaspGLOW fluorescein active caspase staining kits (Biovision, Milpitas, CA, USA) according to the manufacturer’s protocol. The caspase activity was detected using an AccuriTM C5 cytometer.
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