Total cell lysates prepared as in the western blot protocol were used to measure caspase-3/9 activity with caspase-3 or caspase-9 activity kits (C1116 or C1158; Beyotime Institute of Biotechnology). The OD was measured at 405 nm on a microplate reader (Bio-Rad 550; Bio-Rad Laboratories, Inc.) according to the manufacturer’s protocol.
C1158
Manufactured by Beyotime
Sourced in China
The C1158 is a laboratory equipment product. It is used for a specific function within a laboratory setting. No further details about its intended use or capabilities are provided.
Automatically generated - may contain errors
4 protocols using c1158
1
Caspase-3/9 Activity Assay
2
Intestinal Caspase Activities and DAO
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The activities of intestinal Caspase 3 and Caspase 9 were detected by the apoptosis detection kits (Beyotime, C1116 and C1158, Shanghai, China), according to the manufacturer’s instructions. The protein concentrations in the intestine were detected by Bradford method (Beyotime, P0006, Shanghai, China) to normalize the activities of Caspase 3 and Caspase 9. The activity of plasma diamine oxidase (DAO) was detected by the Micro Diamine Oxidase (DAO) Assay Kit (Solarbio, BC1285, Beijing, China), according to the manufacturer’s instruction.
3
Antibody-based Protein Expression Analysis
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The antibodies against ILK (sc-13075), Bcl-2 (sc-492), Bax (sc-493), beta-actin (sc-1616), GAPDH (sc-25778), NF-κB p65 (sc-372), IKB alpha (IKBa; sc-371), and IKBa (ser32; sc-7977) were purchased from Santa Cruz Biotechnology Inc., CA, USA. The antibodies against Histone 1.2 (ab17677) and NF-κB p65 (phospho-S536; ab86299) were purchased from Abcam (Shanghai, China). Kits for measuring caspase-3 (C1116) and caspase-9 (C1158) activities and a lactate dehydrogenase (LDH) assay kit (C0017) were obtained from Beyotime Institute of Biotechnology (Haimen, China). All other reagents were obtained from common commercial sources.
4
Quantifying Caspase-3, -8, and -9 Activities
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Activities of Caspase‐3, ‐8, and ‐9 were assessed using commercial kits (C1116, C1152, C1158; Beyotime) according to the manufacturer's instructions. Briefly, brain tissues were lysed and homogenized. The supernatants of the homogenates were harvested by centrifugation at 12,000 g, and the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (P0012; Beyotime). The lysates were incubated with Ac‐DEVD‐pNA (2 mmol/L, for Caspase‐3 activity), Ac‐IETD‐pNA (2 mmol/L, for Caspase‐8 activity), and Ac‐LEHD‐pNA (2 mmol/L, for Caspase‐9 activity) at 37°C for 2 h. After incubation, absorbance was read at 405 nm using a microplate reader (BioTek). Fold‐increases in activities of Caspase‐3, ‐8, and ‐9 activities were determined by comparison with control (non‐ischemic brain tissue).
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