Cardiomyocyte apoptosis in the area at risk (AAR) has a significant effect on myocardial survival and function. After 3 h of reperfusion, tissue samples from the area at risk (AAR) were analyzed. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl nick-end labeling (TUNEL) and caspase-3 activity assay. TUNEL labeling was performed as described in previous study [6 (link), 20 (link)] by using an in situ cell death detection kit (Roche). In brief, the slides were incubated with TUNEL reaction mixture and then counterstained with the 4′,6-diamino-2-phenylindole (DAPI) to detect the nuclei. The apoptosis index was calculated as a percentage of the number of TUNEL-positive apoptotic cells over the total number of nucleated cells (DAPI staining). Myocardial caspase-3 activity was determined as described before [21 (link)] by using a caspase colorimetric assay kit (Chemicon, Temecula, CA, USA) according to manufacturer’s protocol.
Caspase colorimetric assay kit
Manufactured by Merck Group
Sourced in United States
The Caspase colorimetric assay kit is a laboratory product designed to detect and quantify caspase enzyme activity. It provides a simple and reliable method for measuring the activation of caspase-3, caspase-8, and caspase-9 in cell lysates or purified enzyme preparations.
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6 protocols using caspase colorimetric assay kit
1
Quantifying Cardiomyocyte Apoptosis in Ischemic Heart
2
Assessing Cardiomyocyte Apoptosis with TUNEL and Caspase-3
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DNA fragmentation was detected in situ with the use of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), as described previously [25] . The hearts were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 6 μm thick sections and treated as instructed in the In Situ Cell Death Detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The nuclear density was determined via manual counting of the 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei in five fields for each animal with a×40 objective, and the number of TUNEL-positive nuclei was determined by examining the entire section with the same power objective. The cardiac caspase-3 activity was measured using a caspase colorimetric assay kit following the manufacturer's instructions (Chemicon, Temecula, CA, USA). The absorbance of the pnitroaniline cleaved by caspase was measured at 405 nm using a microplate reader (ELx800; BioTek Instruments, Winooski, VT, USA).
3
Caspase-3 Activity Assay Protocol
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The activity of caspase-3 was determined by Caspase Colorimetric Assay Kit (Sigma-Aldrich Co.) according to the manufacturer’s protocol. Briefly, cells were collected and lyzed in the lysis buffer provided with the kit. Caspase-3 substrate was added to each well. The hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3 results in the release of the p-nitroaniline (pNA) moiety. The chromophore pNA was detected using a spectrophotometer at a wavelength of 405 nm. The caspase enzymatic activity in cell lysates was directly proportional to the color reaction.
4
Quantifying Caspase-3 and Caspase-8 Activities
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Caspase-3 and caspase-8 activities were detected according to the manufacturer’s instructions of the caspase colorimetric assay kit (Sigma). Briefly, cells (108 cell/ml) were collected and resuspended in ice-cold buffer. Following lysis, cell lysates were centrifuged at 12,000 × g for 10 min and the extracts containing 50 μg of protein were incubated with 100 μl of Ac-DEVD-pNA substrate at 37°C for 2 h. The colorimetric release of p-nitroaniline from the Ac-DEVD-pNA substrate was measured at 405 nm.
5
Quantifying Cardiomyocyte Apoptosis after Reperfusion
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After 3 h of reperfusion, terminal deoxynucleotidyl nick-end labeling (TUNEL) assay was used to detect cardiomyocyte apoptosis. TUNEL staining was performed as described previously using in situ cell death detection kits (Roche) [19 (link)]. The apoptosis index was expressed as the number of apoptotic cells (TUNEL-positive staining)/the total number of nucleated cells (4′,6-diamino-2-phenylindole staining) ×100%. Myocardial caspase-3 activity was measured to detect cardiomyocyte apoptosis with the use of caspase colorimetric assay kits (Chemicon, Temecula, CA, USA) as we described before [29 (link)].
6
Evaluating Cardiomyocyte Apoptosis and Oxidative Stress
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After 3 hours of reperfusion, a terminal deoxynucleotidyl nick-end labeling (TUNEL) assay was used to detect cardiomyocyte apoptosis. TUNEL staining was performed as described previously (Wang et al., 2014) using in situ cell death detection kits (Roche).
The apoptosis index was expressed as the number of apoptotic cells (TUNEL-positive staining)/the total number of nucleated cells (4 0 ,6-diamino-2-phenylindole stainingÞÂ 100%. Myocardial caspase-3 activity was measured to detect cardiomyocyte apoptosis with the use of caspase colorimetric assay kits (Chemicon, Temecula, CA) as described before (Fu et al., 2014) .
Quantification of Superoxide Generation, Malonaldialdehyde (MDA) and Superoxide Dismutase (SOD)
Superoxide generation in heart tissue was determined as we described previously (Ding et al., 2015) by lucigenin-enhanced chemiluminescence. The results were indicated as relative light units (RLU) per milligram dry weight per second (RLU/mg/s). The antioxidant enzyme SOD activities and the MDA level in myocardial homogenates were determined spectrophotometrically as described before.
The apoptosis index was expressed as the number of apoptotic cells (TUNEL-positive staining)/the total number of nucleated cells (4 0 ,6-diamino-2-phenylindole stainingÞÂ 100%. Myocardial caspase-3 activity was measured to detect cardiomyocyte apoptosis with the use of caspase colorimetric assay kits (Chemicon, Temecula, CA) as described before (Fu et al., 2014) .
Quantification of Superoxide Generation, Malonaldialdehyde (MDA) and Superoxide Dismutase (SOD)
Superoxide generation in heart tissue was determined as we described previously (Ding et al., 2015) by lucigenin-enhanced chemiluminescence. The results were indicated as relative light units (RLU) per milligram dry weight per second (RLU/mg/s). The antioxidant enzyme SOD activities and the MDA level in myocardial homogenates were determined spectrophotometrically as described before.
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