Caspase 3

Manufactured by Elabscience

Sourced in United States, China

Caspase-3 is a protein-cleaving enzyme that plays a central role in the execution phase of cell apoptosis, or programmed cell death. It is a key mediator of apoptosis in mammalian cells.

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E-EL-R0160/ Caspase-3

8 protocols using caspase 3

Ovarian Oxidative and Inflammatory Biomarkers

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About 0.1 g of the right ovaries of each animal were homogenized on ice in 10 ml of cold phosphate buffer solution. The resultant solutions were centrifuged at 10,000 rpm for15 mins in cold centrifuge after which the supernatant was decanted into tubes and stored at −20°C for biochemical assay later.
The ovarian concentrations of UA [26 (link)], MDA [27 (link)], and GSH [28 (link)] were assayed by colorimetric method as previously reported. The activities of XO [27 (link)], superoxide dismutase (SOD) [28 (link)], catalase [28 (link)], glutathione peroxidase (GPx) [29 (link)], glutathione-S-transferase (GST) [27 (link)], and myeloperoxidase (MPO) [29 (link)] in the ovarian tissues were assayed by colorimetry as earlier documented. Also, the ovarian levels of TNF-α (Elabscience Biotechnology Inc., U.S.A.), IL-6 (Elabscience Biotechnology Inc., U.S.A.), 8OHdG (Elabscience Biotechnology Inc., U.S.A.), and VCAM-1 (Elabscience, Wuhan, China) as well as the activity of caspase 3 (Elabscience Biotechnology Inc., U.S.A.) were determined using ELISA kit following the manufacturers’ guidelines. Ovarian DNA fragmentation index was determined by diphenylamine methods as previously reported [25 ].

Afolabi O.A., Hamed M.A., Anyogu D.C., Adeyemi D.H., Odetayo A.F, & Akhigbe R.E. (2022). Atorvastatin-mediated downregulation of VCAM-1 and XO/UA/caspase 3 signaling averts oxidative damage and apoptosis induced by ovarian ischaemia/reperfusion injury. Redox Report : Communications in Free Radical Research, 27(1), 212-220.

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Biomarkers Assay Protocols for Hepatic and Renal Functions

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AFB₁, thiobarbituric acid (TBA), 2’, 7′-dichlorodihydrofluorescin diacetate (DCFH-DA), 5’, 5’-dithiobis-2-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide (H₂O₂), potassium chloride (KCl), trichloroacetic acid (TCA), sodium azide, glutathione (GSH), epinephrine, sulphosalicylic acid, xanthine, Griess reagent, and O-dianisidine were bought from Sigma-Aldrich Chemical (St. Louis, MO, USA). Biomarkers of hepatic and renal functions such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, and creatinine were purchased from Randox^TM Laboratories Limited, (Crumlin, UK). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin 1-beta (IL-1β), interleukin-10 (IL-10), caspase-9, and caspase-3 were purchased from Elabscience Biotechnology Company (Wuhan, China).

Owumi S.E., Ajakaiye B., Akinwunmi A.O., Nwozo S.O, & Oyelere A.K. (2023). The Hydrophobic Extract of Sorghum bicolor (L. Moench) Enriched in Apigenin-Protected Rats against Aflatoxin B1-Associated Hepatorenal Derangement. Molecules, 28(7), 3013.

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Western Blot Analysis of Protein Expression

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Total protein from the above tumor tissues, MC38 cells and CT26 cells treated with TBMS1 and TEPP-46, RAW264.7 induced by 20 ng/mL IL-4 and treated with TBMS1 was extracted by using RIPA lysis (Millipore, USA) added proteinase and phosphatase inhibitor cocktails (Solarbio, Beijing). The protein concentration was determined with the Pierce BCA detection kit (Thermo Fisher Scientific, USA). The sample with 20-80 µg protein was electrophoresed by 10% polyacrylamide gradient gels at 110 v for 1.5 h and transferred onto 0.45 µm PVDF membrane (Millipore, United States), then TBST containing 5% skim milk was used to block membranes for 2 h. The treated membranes could be incubated with primary antibodies including PKM2 (Huabio, CHN), caspase-3 (Elabscience, CHN), cleaved-caspase-3 (Cell Signaling Technology, USA), GSDME (Abclonal, CHN), GSDME-N (Abcam, UK), STAT6 (Absin, CHN), p-STAT6 (Absin, CHN), β-actin (Huabio, CHN). After incubating overnight at 4°C, the membranes were washed with TBST, and the anti-rabbit or antimouse HRP-conjugated secondary antibody (Bioss, CHN) was incubated with the membranes for 2 h at room temperature. After being washed again by TBST, band detection was visualized by enhanced chemiluminescence reagent (Millipore, USA). Finally, Image J software was used to quantify the integrated density of the target proteins.

Hu D., Cui L., Zhang S., He S., Zhuo Y., Li D., Zhang L., Wang Y., Yang L, & Wang X. (2024). Antitumor effect of tubeimoside-I on murine colorectal cancers through PKM2-dependent pyroptosis and immunomodulation. Naunyn-Schmiedeberg's archives of pharmacology, 397(6).

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Immunohistochemical Analysis of Liver Proteins

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The liver paraffin sections were dewaxed and processed as previously described [34 (link),36 (link)]. Sections were IHC stained using the primary antibodies: rabbit-polyclonal-antibody against NF-kB p65 (1:200), Nrf2 (1:200) (Fisher-Scientific Inc., Waltham, MA, USA), Bcl2 (1:200), and caspase-3 (1:200) (Elabscience Biotechnology Inc., Houston, TX, USA). Diaminobenzidine (DAB) was used for visualization.

Rizq A.T., Sirwi A., El-Agamy D.S., Abdallah H.M., Ibrahim S.R, & Mohamed G.A. (2023). Cepabiflas B and C as Novel Anti-Inflammatory and Anti-Apoptotic Agents against Endotoxin-Induced Acute Kidney and Hepatic Injury in Mice: Impact on Bax/Bcl2 and Nrf2/NF-κB Signalling Pathways. Biology, 12(7), 938.

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Biomarker Analysis of BALF and Lung Tissue

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Total protein (Thermo Fisher Scientific, Waltham, MA, USA) and lactate dehydrogenase (LDH) cytotoxicity (Thermo Fisher Scientific) were determined in BALF. α₁-antitrypsin (Cusabio, Wuhan, Hubei, China), neutrophil elastase (Cusabio), 8-isoprostane (Cayman, Ann Arbor, MI, USA), interleukin (IL)-6 (R&D Systems, Minneapolis, MN, USA), caspase-3 (Elabscience, Houston, TX, USA), ACE2 (Elabscience), AngII receptor type 1 (AT1) (Elabscience) and AngII receptor type 2 (AT2) (Elabscience) were determined in lung lysates using ELISA. All analyses were in accordance with the manufacturers’ instructions. Data obtained from lung lysates are presented after adjusting for the β-actin level (Cell Signaling, Danvers, MA, USA).

Chuang H.C., Chen Y.Y., Hsiao T.C., Chou H.C., Kuo H.P., Feng P.H., Ho S.C., Chen J.K., Chuang K.J, & Lee K.Y. (2020). Alteration in angiotensin-converting enzyme 2 by PM1 during the development of emphysema in rats. ERJ Open Research, 6(4), 00174-2020.

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ELISA Analysis of Inflammatory Markers

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Enzyme-linked immunosorbent assay (ELISA) was used to investigate the tumor necrosis factor (TNF)-α (Invitrogen, Waltham, MA, United States), chemokine (CXC motif) ligand 1/keratinocyte chemoattractant (CXCL1/KC; R&D Systems Inc., Minneapolis, MN, United States), and interleukin (IL)-1β (Invitrogen) in the BALF and serum samples. TNF-α, KC, IL-1β, matrix metallopeptidase (MMP)-9 (R&D Systems), and caspase-3 levels (Elabscience, Houston, Texas, United States) in lung lysates were determined. All of the ELISA kits were conducted following the manufacturers’ instructions. The levels of these markers determined in lung tissues were adjusted based on the total protein in lung lysates. The detailed information of these ELSA kits was shown in Supplementary Table S1.

Chen X.Y., Chen Y.Y., Lin W., Chien C.W., Chen C.H., Wen Y.C., Hsiao T.C, & Chuang H.C. (2020). Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells on the Acute Cigarette Smoke-Induced Pulmonary Inflammation Model. Frontiers in Physiology, 11, 962.

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Histological and Immunohistochemical Analysis of Liver and Kidney Tissues

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For hematoxylin and eosin (H&E) stain, liver and kidney tissues were fixed in neutral buffered formalin, ethanol dehydrated, and paraffin embedded. Paraffin blocks were cut and stained with H&E. Sections were randomly examined in a blind manner and the pathological injuries were graded as previously described [30 (link),31 ,32 (link)].
For IHC, paraffin sections of the liver and kidney were dewaxed and handled as previously demonstrated [30 (link),33 (link)]. IHC was carried out using the primary antibodies: Nrf2 (1:200) (Fisher Scientific Inc., Waltham, MA, USA), rabbit-polyclonal-antibody against NF-κB p65 (1:200), caspase-3 (1:200), and Bcl2 (1:200) (Elabscience Biotechnology Inc., Houston, TX, USA). Diaminobenzidine (DAB) was utilized for visualization.

Alqrad M.A., El-Agamy D.S., Ibrahim S.R., Sirwi A., Abdallah H.M., Abdel-Sattar E., El-Halawany A.M., Elsaed W.M, & Mohamed G.A. (2023). SIRT1/Nrf2/NF-κB Signaling Mediates Anti-Inflammatory and Anti-Apoptotic Activities of Oleanolic Acid in a Mouse Model of Acute Hepatorenal Damage. Medicina, 59(7), 1351.

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Cardioprotective Agents Evaluation Protocol

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HF (CAS: 6665-86-7; H0852; purity ≥ 97%) was purchased from TCI Shanghai Chemical Industry Development Co., Ltd. (Shanghai, China). Diltiazem was purchased from Shanghai Pharmaceutical Co., Ltd. (Lot: H10970375, Shanghai, China). TTC (Lot: G3005) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). AST (Lot: C010-2-1), CK (Lot: A032-1-1), LDH (Lot: A020-2), SOD (Lot: A001-3), and MDA (Lot: A003-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, Nanjing, China). Rat c-TnI (Lot: E-EL-R1253c), IL-1β (Lot: E-EL-R0012c), IL-6 (Lot: E-EL-R0015c), TNF-α (Lot: E-EL-R2856c), and Caspase-3 (Lot: E-EL-R0160c) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The TUNEL assay kit (Lot: 49330900) was purchased from Roche, Switzerland. TRIzol reagent (Cat#RK145), the first-strand cDNA synthesis kit (Cat#RK145), and SYBR green supermix (Cat#RK145) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The anti-p-p38 (Lot. WLP1576), anti-p38 (Lot. WL00764), anti-p-ERK1/2 (Lot. WLP1512), anti-ERK1/2 (Lot. WL01864), anti-p-JNK (Lot. WL01813), anti-JNK (Lot. WL01295), anti-p-NF-κB p65 (Lot. WL02169), anti-NF-κB p65 (Lot. WL01273b) and HRP-conjugated goat anti-rabbit antibody (Lot. WLA023a) were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China).

Zhang Q., Peng Y., Liu J., Yang Y., Hu Z., Zhou Y., Ma J, & Zhang D. (2022). 7-Hydroxyflavone Alleviates Myocardial Ischemia/Reperfusion Injury in Rats by Regulating Inflammation. Molecules, 27(17), 5371.

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