Caspase activation was evaluated using a CaspaTag Caspase 3/7 in situ Assay Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. The green fluorescence signal directly corresponds to the amount of active caspases present in the cells. Stained cells were analyzed using a FACScan flow cytometry platform.
Caspatag caspase 3 7 in situ assay kit
Manufactured by Merck Group
Sourced in United States
The CaspaTag Caspase 3/7 in situ Assay Kit is a laboratory product designed to detect and measure the activity of caspase-3 and caspase-7 enzymes in cells. It provides a fluorescent-based method to visualize and quantify apoptosis or programmed cell death.
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4 protocols using caspatag caspase 3 7 in situ assay kit
1
Caspase 3/7 Activity Assay
2
Caspase-3/7 Activity Quantification
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Cells were harvested and caspase-3/7 activity was examined using a CaspaTag Caspase-3/7 in situ assay kit (Millipore) according to the manufacturer’s instructions. Briefly, 1 x 106 cells were resuspended in 300 μl of medium with different treatments and combined with 10 μl of freshly prepared 30x FAM-DEVD-FMK (FLICA) reagent at 37°C for 1 hr. Caspase-3/7 activities were analyzed by FACS.
3
Apoptosis and Necrosis Quantification in H9c2 Cells
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Apoptosis of H9c2 cells was measured by the presence of active caspase-3/7, detected by flow cytometry (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA) using the CaspaTag Caspase-3/7 In Situ Assay Kit (Chemicon International, Temecula, CA, USA), according to the manufacturer's instructions. Active caspase-3/7 positive cells were assessed on the basis of the fluorescence after incubation with Fluorochrome Inhibitors of Caspases (FLICA) reagent [15 (link)]. 10,000 events were acquired per trial, and the data were expressed as fluorescence intensity in the M2 area of the histogram. The activity of caspase-3/7 was expressed as the mean fluorescence intensity.
Necrosis was assessed via flow cytometric analysis of H9c2 cells incubated with propidium iodide (PI) solution (250 μg/ml) (Millipore, Bedford, MA, USA) for 10 min. PI is membrane impermeable in live cells but binds to DNA of dead cells due to loss of cell membrane integrity, causing increase in the fluorescence. For the analysis, the percentage of PI positive cells was determined using a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) (10,000 events) [16 (link)].
Necrosis was assessed via flow cytometric analysis of H9c2 cells incubated with propidium iodide (PI) solution (250 μg/ml) (Millipore, Bedford, MA, USA) for 10 min. PI is membrane impermeable in live cells but binds to DNA of dead cells due to loss of cell membrane integrity, causing increase in the fluorescence. For the analysis, the percentage of PI positive cells was determined using a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) (10,000 events) [16 (link)].
4
Quantifying Apoptosis in SH-SY5Y Cells
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The Hoechst stain is a cell-permeable fluorescent dye that enters apoptotic cells and binds chromatin DNA for fluorescent detection of apoptosis. SH-SY5Y cells were seeded in a 96-well plate (3 × 104 cells/well) and incubated for 24 h before drug treatment [83 (link)]. Cells were pretreated with 1T3O for 1 h before 6-OHDA (20 μM) was added. The plate was then incubated for 15 h and apoptosis was assessed using the Hoechst stain (CaspaTag™ Caspase 3, 7 In Situ Assay Kit (Chemicon International, Temecula, CA, USA). Briefly, 0.5 μL Hoechst stain (0.5% v/v) was added into the culture media and allowed to react for 5 min. After washing the cells twice with a wash buffer, a fixative was added and apoptosis was evaluated using a fluorescent microscope (Leica, DMI-3000B). The apoptosis ratio was calculated on the basis of the method proposed by Zhao et al. in 2007 [84 (link)]. Five random fluorescent images and five corresponding optical images of the same positions were taken using an inverted fluorescent microscope. The apoptosis ratio was calculated by dividing the number of apoptotic cells by the number of total counted cells. The mean was calculated and taken as the actual apoptotic ratio [84 (link)].
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