HT-29 cells were incubated with A. veronii or E. coli K12 at MOI 1 and 10 for 2 and 4 hours. Levels of active caspase 3/7 and caspase 9 were measured using CellEvent Caspase-3/7 Green ReadyProbes Reagent (Invitrogen) and CaspGLOW Fluorescein Active Caspase-9 Staining Kit (Invitrogen) following the manufacturer’s instructions.
Caspglow fluorescein active caspase 9 staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CaspGLOW Fluorescein Active Caspase-9 Staining Kit is a laboratory instrument designed to detect and measure the activity of caspase-9, a key enzyme involved in the apoptotic signaling pathway. The kit uses a fluorescein-labeled inhibitor of caspase-9 to stain cells, allowing for the identification and quantification of cells undergoing apoptosis.
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Lab products found in correlation
Cellevent caspase 3 7 green readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Casp glow fluorescein active caspase 8 staining kit, by Thermo Fisher Scientific (1 mentions)
Novocyte 2060 flow cytometer, by Agilent Technologies (1 mentions)
Vybrant fam caspase 8 assay kit, by Thermo Fisher Scientific (1 mentions)
12 well plate, by Corning (1 mentions)
6 protocols using caspglow fluorescein active caspase 9 staining kit
1
Apoptosis Induction in HT-29 Cells
2
Quantifying Apoptosis via Caspase-9 Assay
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Cells were analyzed using CaspGLOW Fluorescein Active Caspase-9 Staining Kit (Invitrogen, Carlsbad, CA, USA). The caspase-9 inhibitor LEHD-fluoromethylketone conjugated to FITC (FITC-LEHD-FMK) irreversibly binds to active caspase-9 in apoptotic cells. Briefly, T cells in presence of ADV or vehicle control were centrifuged for 5 min at 400g and the supernatant was removed by aspiration. The cell pellet was resuspended in 200 μl of PBS, containing FITC-LEHD-FMK at a dilution of 1:500, and the cells were incubated for 30 min at 37°C. Afterward, the cell suspension was centrifuged, the supernatant was removed, and the cells were washed twice in 300 μl of wash buffer. After the final washing step, cells were resuspended in 200 μl of wash buffer, and analyzed using flow cytometry (FACS Calibur, BD Biosciences).
3
Apoptosis Analysis via Caspase-8 and Caspase-9
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Cells were analyzed by using CaspGLOW Fluorescein Active Caspase-8 Staining Kit and CaspGLOW Fluorescein Active Caspase-9 Staining Kit (Invitrogen, Carlsbad, CA, USA). The caspase-8 inhibitor IETD-fluoromethylketone conjugated to FITC (FITC-IETD-FMK), and caspase-9 inhibitor LEHD-fluoromethylketone conjugated to FITC (FITC-LEHD-FMK) were irreversibly bound to active caspase-8 or active caspase-9 in apoptotic cells, respectively. Briefly, T cells in the presence of Pitavastatin or vehicle controls were centrifuged for 5 min at 400× g, and the supernatant was removed by aspiration. The cell pellet was resuspended in 200 μL of PBS, containing FITC-IETD-FMK or FITC-LEHD-FMK at a dilution of 1:500, and the cells were incubated for 30 min at 37 °C. Afterwards, the cell suspension was centrifuged, the supernatant was removed and the cells were washed twice with 300 μL wash buffer. After the final washing step, cells were resuspended in 200 μL wash buffer and analyzed by using flow cytometry (FACS Calibur, BD Biosciences, Franklin Lakes, NJ, USA). The data of at least three independent experiments were analyzed by using the FlowJo software.
4
Apoptosis Induction in HT-29 Cells
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HT-29 cells were incubated with A. veronii or E. coli K12 at MOI 1 and 10 for 2 and 4 hours. Levels of active caspase 3/7 and caspase 9 were measured using CellEvent Caspase-3/7 Green ReadyProbes Reagent (Invitrogen) and CaspGLOW Fluorescein Active Caspase-9 Staining Kit (Invitrogen) following the manufacturer’s instructions.
5
Flow Cytometric Analysis of Apoptotic Caspase Activation
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For flow cytometry detection of activated caspase-8 and caspase-9 in apoptotic cells, HEK293 and MCF-7 were cultured in 12-well plates (Corning Inc., Gllendale, AZ, USA) (5 × 105 cells/well) in DMEM, containing 10% FBS for 24 h. Cells were treated with compound 29 (at appropriate IC50 concentration) for various time points. After incubation with the compound, 29 cells were harvested and stained with Vybrant FAM Caspase-8 Assay Kit (# V35119, Thermo Fisher Scientific, Waltham, MA USA) and CaspGLOW Fluorescein Active Caspase-9 Staining Kit (#88-7006-42, Thermo Fisher Scientific, Waltham, MA USA) according to the manufacturer’s recommendations. The samples were analyzed by NovoCyte 2060 flow cytometer (Agilent Technologies, Inc., Santa Clara, CA, USA), using 488 nm excitation and collecting fluorescence emission; a 530/30 bandpass filter for caspase reagent, and a 690/50 BP filter for propidium iodide (PI) dead cell staining. Following the application of the standard fluorescence compensation technique, medians of fluorescence histogram into caspase+/PI− cell populations were used for statistical analysis (15,000 events were collected in each probe gated as “live cells”).
6
Caspase-9 Activity in Daunorubicin-Treated THP-1 Cells
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Caspase-9 activity in daunorubicin-treated THP-1 cells was measured using the CaspGLOW Fluorescein Active Caspase-9 Staining Kit (Thermo Fisher Scientific). Approximately 5 × 105 cells in 0.3 mL IMDM + 10% FBS were stained with 1 µL FITC-LEHD-FMK and incubated for 30 min in a CO2 incubator before washing twice with the supplied wash medium and analysis with flow cytometry. Both untreated and treated, but not stained THP-1 cells, were used as negative controls. At least 25,000 gated cells were collected.
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