Caspase 9

Manufactured by Bioss Antibodies

Sourced in United States, China

Caspase-9 is a key enzyme involved in the initiation of the apoptotic pathway. It plays a central role in the activation of other caspases, leading to programmed cell death. Caspase-9 is an important tool for researchers studying apoptosis and cell death mechanisms.

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Lab products found in correlation

Caspase 3, by Bioss Antibodies (3 mentions) Fluorescence microscope, by Olympus (1 mentions) Bmi 1, by Santa Cruz Biotechnology (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Survivin, by Santa Cruz Biotechnology (1 mentions) Cox 4, by Abbkine (1 mentions) C myc, by Santa Cruz Biotechnology (1 mentions) Bcl xl, by Santa Cruz Biotechnology (1 mentions) Cyclind1, by Immunoway (1 mentions) Protein phosphatase inhibitor, by Solarbio (1 mentions) Gapdh, by Wuhan Servicebio Technology (1 mentions) Ripa buffer, by Solarbio (1 mentions) Protease inhibitor mixture, by Solarbio (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Bcl2 associated x (bax), by Proteintech (1 mentions) Sirt1, by Bioss Antibodies (1 mentions) Vascular endothelial growth factor (vegf), by Bioss Antibodies (1 mentions) Vimentin, by Abcam (1 mentions) Gel doc 2000, by Bio-Rad (1 mentions) Bca protein quantification kit, by Beyotime (1 mentions)

Spelling variants of this product

Anti-caspase-9 (3 citations)

7 protocols using caspase 9

Apoptosis Evaluation of HUCMSCs with Calcium Hydroxide

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HUCMSCs were assigned into 9 groups of control and 9 groups of calcium hydroxide treatment, with allocation for 1, 3, and 7 days of observations. Each group consisted of six wells of M24 plate (Iwaki Asahi, Japan). Every M24 plate well was seeded with 250.000 HUCMSCs in 1 mL media. HUCMSCs in the treatment groups were grown in minimum essential medium (MEM) alpha containing 0.1 microgram/mL calcium hydroxide, and the control groups were grown in MEM alpha medium only without the addition of calcium hydroxide. The groups were cultured in incubator at 37°C and 5% CO₂ and observed for 1, 3, and 7 days.
Apoptosis reaction of HUCMSCs was investigated through the expressions of apoptotic protease-activating factor-1 (Bioss Antibodies, USA), Caspase-3 (Bioss Antibodies, USA), and Caspase-9 (Bioss Antibodies, USA). The investigation was done following the manufacturer’s instructions Other previous study used flow cytometric analysis for assessments.12 (link) In this research APAF-1, Caspase-3, and Caspase-9 expressions were assessed for apoptosis using polyclonal antibody with fluorescence isothiocyanate (FITC) label (Bioss Antibodies, USA). Observation of the results was done with fluorescence microscope (Olympus, Japan) with imaging system at 100x magnification and processed in ImageJ software for fluorescence quantification (National Institute of Health, USA).13 (link)

Prasetyo E.P., Kuntjoro M., Goenharto S., Juniarti D.E., Cahyani F., Hendrijantini N., Nugraha A.P., Hariyani N, & Rantam F.A. (2021). Calcium Hydroxide Increases Human Umbilical Cord Mesenchymal Stem Cells Expressions of Apoptotic Protease-Activating Factor-1, Caspase-3 and Caspase-9. Clinical, Cosmetic and Investigational Dentistry, 13, 59-65.

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Protein Extraction and Western Blot Analysis

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The total protein was extracted using a RIPA lysis buffer (Beyotime). The cytosolic protein, mitochondrial protein and nuclear protein were extracted respectively using a mitochondria and nuclear protein extraction kit (Beyotime) according to the manufacturer's instructions. Equal amounts of proteins were loaded onto SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non- fat milk powder in TBST for 1 h and incubated with primary antibodies against Bmi1 (1:500) (Santa Cruz), Bcl-2 (1:500) (CST), Bax (1: 1000) (CST), c-Myc (1:400) (Santa Cruz), Cyclin D1 (1:500) (CST), survivin (1:400) (Santa Cruz), Bcl-xL (1:400) (Santa Cruz), caspase 3 (1:1000) (CST), caspase 9 (1:300) (Bioss), COX IV (1:1000) (Abbkine). PARP-1 (1:1000), cytochrome-C (1:1000), Histone H3 (1:2000), phosphorylated IKKα/β (1:1000), IKKβ (1:1000), phosphorylated IκBα (1:1000), IκBα (1:1000), NF-κB/p65 (1:1000), GAPDH (1:1000), β-actin (1:1000) were purchased from CST. After washing with TBST, the membrane was incubated with secondary, horseradish peroxidase-coupled antibodies (Pierce) and visualized using enhanced chemiluminescence (Pierce). GAPDH or β-actin was used as internal controls.

Yin T., Zhang Z., Cao B., Duan Q., Shi P., Zhao H., Camara S.N., Shen Q, & Wang C. (2016). Bmi1 inhibition enhances the sensitivity of pancreatic cancer cells to gemcitabine. Oncotarget, 7(24), 37192-37204.

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Apoptosis and Cell Cycle Regulation

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Cells were treated with the above concentrations of FB₁ for 48 h, then washed three times with ice-cold PBS. The cells were then lysed with RIPA buffer (Solarbio) containing a protease inhibitor mixture (Solarbio, Beijing, China) and protein phosphatase inhibitor (Solarbio). The total protein concentrations were determined using a Bradford protein kit (Tiandz, Beijing, China). The total proteins were resolved by electrophoresis and transferred to poly(vinylidene fluoride) membranes for 80 min at 200 mA. PVDF membranes were incubated with primary antibodies caspase3 (Beijing Biosynthesis Biotechnology Co., Ltd. Beijing, China), caspase9 (Bioss), CDK2 (Bioss), CDK4 (Bioss, Beijing, China), cyclinD1 (immunoway, Plano, TX 75024, USA), cyclinE1 (immunoway), Bax (Proteintech Group, Inc., Wuhan, China), and GAPDH (Servicebio, Wuhan, China) overnight at 4 °C. Then, the membrane was washed three times with Tris-buffered saline tween (TBST) for 15 min. Secondary antibody (IgG, Vazyme) was used to incubate the membranes for 1 h at 4 °C. Finally, band density was quantified using the Image Lab 5.0 on the ChemiDOC XRS + system (Bio-Rad, Hercules, CA, USA).

Wang T., Lei H., Zhou L., Tang M., Liu Q., Long F., Li Q, & Su J. (2022). Effect of Fumonisin B1 on Proliferation and Apoptosis of Intestinal Porcine Epithelial Cells. Toxins, 14(7), 471.

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Immunohistochemical Analysis of Cartilage Markers

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Tissue specimens were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Serial 4 μm sections were obtained. The tissue sections were dewaxed, hydrated, and treated with pepsin, whereupon they were washed with PBS, treated with peroxidase blocker, and washed again with PBS. The sections were incubated with SIRT1, VEGF, PTEN, AKT, Caspase 9, MMP13, and type II collagen antibodies (Bioss, Beijing, China). They were then washed with PBS, incubated with the secondary antibody, and then treated with fresh Diaminobenzidine (DAB) solution. Tissues that were found to be immunohistochemically positive for SIRT1, VEGF, PTEN, AKT, Caspase 9, MMP13, and type II collagen proteins were stained brown under a microscope. The area was photographed under the positive fluorescence microscope, and an area of the same size was intercepted. Image Pro-Plus 6.0 was used to calculate the size of the brown area.

Yu F., Li M., Yuan Z., Rao F., Fang X., Jiang B., Wen Y, & Zhang P. (2018). Mechanism research on a bioactive resveratrol– PLA–gelatin porous nano-scaffold in promoting the repair of cartilage defect. International Journal of Nanomedicine, 13, 7845-7858.

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Protein Expression Analysis in Lysed Cells

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The treated cells were lysed with RIPA (Beyotime, Shanghai, China) containing protease inhibitors. The protein concentration was determined with the BCA protein quantification kit (Beyotime, China). After that, the protein was separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. PVDF membranes were closed with 5% skim milk powder for 2 h at room temperature, and the primary antibodies CD206 (1:1000; CST), CD163 (1:1000; Abcam), CD68 (1:1000; Abcam), CD86 (1:1000; Bioss), CD63 (1:500; Abcam), CD9 (1:500; Abcam), Caspase-3 (1:750; Bioss), Caspase-9 (1:750; Bioss), MMP-2 (1:1000; HUABIO), MMP-9 (1:1000; HUABIO), E-cadherin (1:1000; Abcam), Vimentin (1:1000; Abcam), and PTEN (1:1000; Abcam) were incubated at 4 °C overnight. After incubation of the secondary antibody for 1 h at room temperature, ECL reagent (Beyotime, Shanghai, China) was added and the bands were observed by Bio-Rad GelDoc 2000.

Li J., Bao Y., Peng S., Jiang C., Zhu L., Zou S., Xu J, & Li Y. (2023). M2 Macrophages-Derived Exosomal miRNA-23a-3p Promotes the Progression of Oral Squamous Cell Carcinoma by Targeting PTEN. Current Issues in Molecular Biology, 45(6), 4936-4947.

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Hepatocyte Protein Extraction and Western Blot

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Hepatocyte protein was isolated using a protein extraction kit (BestBio Biotech Co. Ltd., Shanghai, China), and the BCA protein assay kit (BestBio) was used to determine the concentration of protein samples.
Western blotting was performed with the method described by [47] with the following primary antibodies: anti-PPARα (Abcam, Cambridge, UK), anti-PCNA (ABclonal Technology, Wuhan, China), anti-CDK2 (Zen-Bio, Chengdu, Chin), anti-caspase-3 and caspase-8 (Abcam), and caspase-9 (Bioss, Beijing, China), anti-PI3K [Cell Signaling Technology (CST), USA], anti-p-PI3K (CST), anti-AKT and p-AKT (CST), and anti-Gsk3β and p-Gsk3-β (ABclonal). Goat anti-mouse and goat anti-rabbit secondary antibodies (Zen-Bio) β-Tubulin (Zen-Bio) was used as a reference.

Cui Z., Jin N., Amevor F.K., Li L., Shu G., Du X., Kang X., Xu D., Ning Z., Deng X., Song W., Tian Y., Zhu Q., Wang Y., Li D., Zhang Y., Wang X., Han X., Feng J., & Zhao X. (2021). Salidroside Alleviates Lipid Metabolism Disorder and Inflammatory Response, and Promotes Hepatocyte Proliferation in Fatty Liver-Hemorrhagic Syndrome of Laying Hens.

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Protective Effects of HDAX on ER Stress

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HDAX were mainly composed of Coptidis Rhizoma, Radix Rehmanniae, Radix Puerariae, Radix Ophiopogonis, Eriobotryae Folium, and Radix Notoginseng, which were purchased from Anhui Provincial Hospital of Chinese Medicine. Notoginsenoside R1, ginsenoside Rg1, puerarin, acteoside, berberine, coptisine, and palmatine hydrochloride were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). 4-phenylbutyric acid (4-PBA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibody of GRP78, CHOP was purchased from A nity Bioscience (1:1300, USA); Caspase-12, Caspase-9, Caspase-3 were obtained from Bioss (1:1000, Beijing, China); Bax, Bcl-2 was provided by Bioworld (1:1000, Nanjing, China).

Ye T., Xuan W., Zhou P., Shao N., Song H., Wang Y., Hu G., Wang L., Wang G., Fang Z., & Ye T. (2021). Huangdi Anxiao Attenuated High Glucose-Induced PC12 Cells Neurotoxicity via Inhibiting Apoptosis Pathway of Endoplasmic Reticulum Stress.

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