DDP was purchased from hansoh pharmaceutical, Jiangsu. MTT, DMSO, Hoechst 33258 solution, BCA protein kit, PI, AO and ECL system (BeyoECL plus) were purchased from Beyotime Biotechnology, China. Rabbit anti-FSHR antibody was obtained from Abcam, USA. Rabbit anti-Bax, Bcl-2, Caspase-3, Caspase-8, Bad, LC3 and mouse anti-β-Actin were purchased from Beyotime Biotechnology, China. Annexin V-FITC / PI double staining apoptosis kit was from Roche, Germany and Novex NuPAGE Gel Electrophoresis Systems was from Life Technologies, USA. And male BALB/c nude mice were purchased from Cavens Lab Animal, Changzhou, China.
Caspase 8
Manufactured by Beyotime
Sourced in China
Caspase-8 is a member of the cysteine-aspartic acid protease (caspase) family. It plays a crucial role in the initiation of the apoptosis (programmed cell death) signaling pathway. Caspase-8 is responsible for cleaving and activating other downstream caspases, leading to a cascade of events that ultimately result in cell death.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Beyotime (5 mentions)
Bcl 2, by Beyotime (2 mentions)
Caspase 9, by Beyotime (2 mentions)
Rabbit anti bax, by Beyotime (1 mentions)
Mouse anti β actin, by Beyotime (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
Dimethyl sulfoxide (dmso), by Beyotime (1 mentions)
Bca protein kit, by Beyotime (1 mentions)
Hoechst 33258 solution, by Beyotime (1 mentions)
P chk1, by Cell Signaling Technology (1 mentions)
P chk2, by Cell Signaling Technology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
P atm, by Cell Signaling Technology (1 mentions)
P brca1, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection kit, by Geneflow (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Agei hf, by New England Biolabs (1 mentions)
7 protocols using caspase 8
1
Apoptosis Assay in FSHR-Expressing Cells
2
Evaluation of Apoptosis and DNA Damage
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CD34+CD38− KG1α cells (5 × 105/ml) were cultured for 24 or 48 h in the absence or presence of 40 nM IDA and 0.75 μM chidamide. The protein expression levels were determined by staining with primary antibodies and relevant HRP-conjugated secondary (1:10,000, Abcam, Cambridge, UK) antibodies. The primary antibodies (caspase-3, caspase-8, caspase-9, and PARP—Beyotime, China; p-BRCA1, p-ATM, p-CHK1, p-CHK2, γH2A.X, and Ace-H3—Cell Signaling, Herts, UK) were diluted at 1:1000 in 5% fat-free milk-TBST. Anti-β-actin (1:1000, Cell Signaling, Herts, UK) was used as a loading control. The signal was detected using an ECL Western Blotting Detection Kit (GeneFlow, Staffordshire, UK).
3
Apoptosis Assessment in Cell Culture
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Cell culture products were provided by Hyclone (Logan, UT). Puromycin, streptomycin and penicillin were provided by Life Technologies, Inc. (Gaithersburg, MD). AO-EB, Caspase-8 and Caspase-3 fluorometric assay kits were provided by Beyotime Biotech (Jiangsu, China). Caspase-8 inhibitor (Z-IETD-FMK) were from MedChem Epress (Princeton, NJ). RT reagent Kit and qRT-PCR kits were from Takara Bio (Dalian, China). FuGENE®HD Transfection Reagent was from Promega Corporation (Madison, WI). Restriction enzymes NcoI, AgeI-HF and EcoRI-HF were from New England Biolabs (Ipswich, Ma). Lactobacillus plantarum NCU116, E. coli Dh5α and pLKO.pig-puro vector were stored at −80 °C.
4
Western Blot Analysis of Cardiac Markers
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The heart tissue and H9C2 cells were ground and lysed by protein lysis buffer (Beyotime, China) with PMSF and phosphatase inhibitor cocktail. Total protein samples were extracted from the lysate, and the protein concentration was detected. The sample was loaded into 8% or 12% SDS-PAGE gel for electrophoresis and then transferred to the PVDF membrane. It was blocked for 1 hour and then immersed in primary antibody for 12 hours at 4°C, such as GAPDH (Proteintech, 60004-1-Ig, USA), GSDMD (Abcam, ab219800, ab209845, UK), NLRP3 (Abcam, ab214185, UK), caspase-1 (Abcam, ab179515, UK), IL-1β (Abcam, ab9722, UK), caspase-3 (Cell Signaling Technology, 14220T, USA), caspase-7 (Cell Signaling Technology, 12827T), caspase-8 (Beyotime, AC056, China), Bcl-2 (Beyotime, AB112, China), Bax (Beyotime, AB026, China), PARP-1 (Cell Signaling Technology, 9532S, USA), and poly/mono-ADP ribose (Cell Signaling Technology, 83732S, USA). The membranes were washed with TBST twice, immersed in the secondary antibody for 1 hour, and then washed again. Bands were displayed by the ECL detection kit (Millipore, USA).
5
Molecular Mechanisms of Cell Death Pathways
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The pan-caspase inhibitor Z-VAD-FMK (S7023) and RIPK1 inhibitor necrostatin-1 (S8037) were purchased from Selleck Chemicals. TNF-α (RP0080S) was the product of Kingfisher Biotech. Staurosporine (S1882) and SM-164 (SC0114) were obtained from Beyotime. The primary antibodies used in this study were as follows: rabbit polyclonal Caspase-3 (AC030; Beyotime), rabbit polyclonal Caspase-8 (AC056; Beyotime), rabbit polyclonal RIPK3 (AF7893; Beyotime), rabbit polyclonal MLKL (A5579; ABclonal), rabbit polyclonal TNF-α (A0277; ABclonal), rabbit polyclonal FasL (A0234; ABclonal), rabbit polyclonal TraiL (A12312; ABclonal), mouse monoclonal GAPDH (AG019; Beyotime), rabbit monoclonal (EPR9627) RIP3 (phospho S227) (ab209384; Abcam), rabbit monoclonal (EPR9514) to MLKL (phospho S358) (ab187091; Abcam), and mouse monoclonal anti-PRV gE (kindly provided by Dr. Gaiping Zhang, College of Veterinary Medicine, Henan Agricultural University, China). The secondary antibodies used for Western blot were HRP-conjugated goat anti-mouse IgG (BS12478; Bioworld Technology) and HRP-conjugated goat anti-rabbit IgG (BS13278; Bioworld Technology).
6
Measuring Caspase Enzyme Activities
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Caspase activities were measured using Caspase-3, Caspase-8 and Caspase-9 (cat nos. C1115, C1151; C1157; Beyotime Institute of Biotechnology) Activity assay kits according to the manufacturers' instructions. Cells were washed with PBS, centrifuged at 4°C (530 × g, 5 min) and 100 µl lysate was used per 2×105 cells and incubated on ice for 15 min. Centrifugation at 4,246 × g at 4°C for 10 min. Protein concentration in supernatant was measured using a Bradford assay kit (Tiangen Biotech Co., Ltd.). Then 50 µl cell lysate supernatant and 10 µl AC-DevD-PNA (2 mM) for Caspase-3, AC-IETD-PNA (2 mM) for Caspase-8 and AC-LEHD -pNA (2 mM) for Caspase-9 was mixed in a 40 µl detection buffer at 37°C for 4 h and then analyzed using a microplate reader (Tecan Group, Ltd.).
7
Caspase Activity Assay Protocol
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