Tissues from the left hemisphere were homogenized separately in 50 mM potassium orthophosphate, pH 7.0, containing 1 mM EDTA. The homogenates were incubated in RIPA buffer for 1 h at 4 °C, and then the samples were centrifuged for 10 min at 10,000 × g at 4 °C. After centrifugation, the lysates were transferred to a new cooled 1.5-ml polyethylene tube. The protein concentration (Bradford method) was measured, and the samples were frozen at − 80 °C for further determination. Caspase-9 activity was determined by an immunoenzymatic method (ELISA (enzyme-linked immunosorbent assay)) using a Caspase-9 Assay Kit (fluorometric, Abcam) according to the manufacturer’s instructions.
Caspase 9 assay kit
Manufactured by Abcam
Sourced in United States
The Caspase-9 Assay Kit is a laboratory tool used to detect and quantify the activity of Caspase-9, an enzyme involved in the initiation of the apoptotic (programmed cell death) pathway. The kit provides the necessary reagents and protocols to measure Caspase-9 activity in cell lysates or purified enzyme samples.
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Lab products found in correlation
Caspase 3 assay kit, by Abcam (2 mentions)
Versafluor fluorometer, by Bio-Rad (1 mentions)
Cathepsin b assay kit, by Abcam (1 mentions)
96 well cell culture plate, by Greiner (1 mentions)
Synergy neo2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Thiazolyl blue tetrazolium bromide mtt assay, by Merck Group (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
Spectramax 340pc plate reader, by Molecular Devices (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Curcumin, by Merck Group (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
Epichlorohydrin ep, by Sinopharm (1 mentions)
Sodium hydroxide (naoh), by Nanjing Chemical Reagent (1 mentions)
Ethylene glycol, by Sinopharm (1 mentions)
Caspase 3 assay kit ab39401, by Abcam (1 mentions)
Etoposide, by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Sense microplate reader, by Hidex (1 mentions)
Caspase 8 assay kit, by Abcam (1 mentions)
13 protocols using caspase 9 assay kit
1
Caspase-9 Activity Determination
2
Caspase and Cathepsin Enzymatic Assays
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Assays of caspase-9 and caspase-3 activity were carried out by using caspase-9 assay kit (abcam, ab65607) and caspase-3 assay kit (abcam, ab39383) according to the manufacturer's protocol as described previously [40 (link)].
Assays of cathepsin B (Cat-B) and cathepsin D (Cat-D) activity were carried out by using cathepsin B assay kit (abcam, ab65300) and cathepsin D assay kit (abcam, ab65302) according to the manufacturer's protocol. Briefly, 50 μg of cytosolic fraction protein prepared from control or stimulated cells was added up to the final reactive to 200 μL per well in a 96-well plate. Aliquots of assay volume were treated with 140 mM site-specific substrates in assay buffer at 37°C with 10 mM DTT for 0.5 h. Cleavage of the preferred Cat-B substrate [sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin)] and Cat-D substrate [sequence GKPILFFRLK(Dnp)-D-R-NH2, labeled with MCA] by Cat-B and Cat-D release AFC and MCA respectively. The AFC fluorescence is measured at 400 nm excitation and 505 nm emission, and the MCA fluorescence is measured at 328 nm excitation 460 nm emission with a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) respectively. The relative fluorescent units (RFU) were normalized with protein concentrations.
Assays of cathepsin B (Cat-B) and cathepsin D (Cat-D) activity were carried out by using cathepsin B assay kit (abcam, ab65300) and cathepsin D assay kit (abcam, ab65302) according to the manufacturer's protocol. Briefly, 50 μg of cytosolic fraction protein prepared from control or stimulated cells was added up to the final reactive to 200 μL per well in a 96-well plate. Aliquots of assay volume were treated with 140 mM site-specific substrates in assay buffer at 37°C with 10 mM DTT for 0.5 h. Cleavage of the preferred Cat-B substrate [sequence RR labeled with AFC (amino-4-trifluoromethyl coumarin)] and Cat-D substrate [sequence GKPILFFRLK(Dnp)-D-R-NH2, labeled with MCA] by Cat-B and Cat-D release AFC and MCA respectively. The AFC fluorescence is measured at 400 nm excitation and 505 nm emission, and the MCA fluorescence is measured at 328 nm excitation 460 nm emission with a VersaFluor Fluorometer (Bio-Rad, Hercules, CA) respectively. The relative fluorescent units (RFU) were normalized with protein concentrations.
3
Caspase-9 Apoptosis Assay Protocol
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To assess apoptosis, 1000 cells /well were seeded in 96‐well cell culture plates (Greiner) for 24 h prior to MP‐470 exposure for 72 h prior to the assay, a Caspase‐9 assay kit (Abcam, cat# ab65607) was then used according to manufacturer's instructions. Fluorescence was then read at 400 nm excitation/505 nm emission using a Synergy Neo2 Multi‐Mode Microplate Reader (BioTek). And 5 μM Staurosporine was used a positive control for apoptosis. The results were averaged over three different independent experiments with triplicate wells measured per experiment.
4
Inducing Apoptosis in TNBC Cells
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Activating human Trail R2/TNFRSF10B (DR5) antibody (Cat # MAB631) from R&D Systems was used to induce apoptosis in TNBC cells. Anti-DR5 and matching non-specific control IgG (R&D Systems) were immobilized overnight at 4°C onto 96-well cell culture plates at 10 μg/mL or 20 μg/mL in DMEM supplemented with 0.5% FBS. Cells were then seeded in DMEM medium containing 0.5% FBS and incubated at 37°C for 24 or 36 h. The resazurin assay was used according to the manufacturer's instructions to assess cell survival and the fluorescence intensity was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). Alternatively, cell survival was monitored using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay (Sigma- Aldrich). The MTT assay was performed according to manufacturer's instructions using a Spectramax 340PC plate reader (Molecular Devices).
To analyse CASPASE-3 or CASPASE-9 activation, 6-well plates were pre-coated with activating anti-DR5 or a matching non-specific IgG as described above. Cells were seeded onto the pre-coated 6-well plates (1 × 106 cells/well) in DMEM medium containing 0.5% FBS and incubated at 37°C for 5 h. Caspase activity was monitored using the EnzChek® CASPASE-3 Assay Kit #1 (Cat # E-13183) or the CASPASE-9 Assay Kit, Cat# ab65608 (Abcam, Toronto, ON, Canada) by following manufacturer's instructions.
To analyse CASPASE-3 or CASPASE-9 activation, 6-well plates were pre-coated with activating anti-DR5 or a matching non-specific IgG as described above. Cells were seeded onto the pre-coated 6-well plates (1 × 106 cells/well) in DMEM medium containing 0.5% FBS and incubated at 37°C for 5 h. Caspase activity was monitored using the EnzChek® CASPASE-3 Assay Kit #1 (Cat # E-13183) or the CASPASE-9 Assay Kit, Cat# ab65608 (Abcam, Toronto, ON, Canada) by following manufacturer's instructions.
5
Caspase 4/9 Activity in TLR-Deficient B Cells
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Splenocyte B cells were separated from WT, TLR2KO and TLR4KO mice and cultured 48 hours with P. gingivalis LPS (10μg/ml), P. gingivalis LPS (10μg/ml) + CpG (10μM) and untreated control. Casp4 and Casp9 protein activities were performed by using Caspase 4 Assay kit (Abcam) or Caspase 9 Assay kit (Abcam) following user’s instruction. Briefly, cells (1×106 per sample) were lysis in 50 μl cell lysis buffer incubated on ice for 10 minutes and then incubated with 50 μl reaction buffer and 5 μl LEHD-AFC substrate at 37°C for 2 hours. The plate was read in a microplate fluorometer reader (BioTek) and fold-increase in Caspase 4/9 activity was determined by comparing these results with the level of the untreated control.
6
In vitro Caspase 9 Activity Assay
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In vitro analysis of caspase 9 activity in HDFs was performed using the Caspase 9 Assay Kit (ab65608, Abcam, US) according to the manufacture’s protocol. Caspase 9 activity was measured based on the absorbance at 400–405 nm, collected using a Spectramax i3x. Data were compared with the absorbance of control samples.
7
Curcumin and Antioxidant Assay Protocol
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Curcumin, ABTS (2,2′-azinobis-3-ethylbenzothiazolin-6-sulfonic acid), and DPPH (1,1′-diphenyl-2-picryhydrazyl) were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). β-CD (chemical purity) was purchased from Shangdong Xinda Biotechnology Co., Ltd. (Zibo, China). Epichlorohydrin (EP) and ethylene glycol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RPMI 1640 medium were purchased from Hyclone Company (South Logan, UT, USA). Fetal bovine serum (FBS) and the antibiotic mixture (penicillin-streptomycin) were purchased from Gibco Company (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Company (Kumamoto, Japan). Caspase 3 assay kit (ab39401), Caspase 8 assay kit (ab39700), and Caspase 9 assay kit (ab65608) were purchased from Abcam Company (Cambridge, UK). Annexin V/PI apoptosis kit were purchased from MultiScience Company (Hangzhou, China). Sodium hydroxide and other reagents were of analytical grade and were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Double distilled and sterilized water was used to prepare all solutions.
8
Quantification of Caspase-8 and Caspase-9 Activity
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HUVECs treated with 250 nM TS5-p45 for 24 h, 10 μg/ml Etoposide (ETOP; Sigma Aldrich, E1383) for 24 h and 0.5 μM Staurosporine (STS; Sigma Aldrich, S6942) for 6 h, respectively, were harvested for isolation of total protein lysate. Protein concentrations were quantified by Bradford assay. Colorimetric Cas-8 and Cas-9 activity measurements were performed with Caspase-8 Assay Kit (Abcam, ab39700) and Caspase-9 Assay Kit (Abcam, ab65608) according to the manufacturer’s protocol. Protein lysate at 50 μg/well was incubated with Cas-8 substrate (IETD-pNA) and Cas-9 substrate (LEHD-pNA), respectively, at 37 °C overnight. The plate was read at 405 nm with Hidex Sense Microplate Reader (Hidex, Finland), and buffer-related background readings were subtracted from sample readings.
9
Quantifying Apoptosis and Caspase-9 Activity
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Flow cytometry was used to analyze cell apoptosis and Annexin V-propidium iodide (AV-PI) staining was performed. Briefly, after treatment cells were harvested and washed three times with PBS. Following centrifugation for 10 min, cells were resuspended in 500 µl of binding buffer including 5 µl FITC-conjugated Annexin V, the mixture was incubated in the dark for 10 min, and then 5 µl of PI was added. Ultimately, all specimens were assessed by flow cytometry with a FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA, USA), and all the results are shown as a percentage of total cells, to quantitatively evaluate the rate of apoptosis.
Caspase-9 activity. The caspase-9 activity was assayed using the Caspase-9 Assay kit (Abcam), according to the manufacture's instruction. The fresh protein lysates from cells were prepared using cell lysis buffer. Then, 85 µl of reaction buffer and 5 µl of LEHD-pNA (Leu-Glu-His-Asp-p-nitroanilide) were added to each sample and incubated at 37˚C for 2 h. The absorbance was measured in an ELISA reader (Labsystems, Helsinki, Finland) at 405 nm.
Statistical analysis. The SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA) was used to analyze the related data with χ 2 or t-tests. The results were considered to indicate a statistically significant result at P<0.05.
Caspase-9 activity. The caspase-9 activity was assayed using the Caspase-9 Assay kit (Abcam), according to the manufacture's instruction. The fresh protein lysates from cells were prepared using cell lysis buffer. Then, 85 µl of reaction buffer and 5 µl of LEHD-pNA (Leu-Glu-His-Asp-p-nitroanilide) were added to each sample and incubated at 37˚C for 2 h. The absorbance was measured in an ELISA reader (Labsystems, Helsinki, Finland) at 405 nm.
Statistical analysis. The SPSS version 19.0 software (SPSS, Inc., Chicago, IL, USA) was used to analyze the related data with χ 2 or t-tests. The results were considered to indicate a statistically significant result at P<0.05.
10
Caspase-9 Activity Assay in H2O2-Treated Cells
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Caspase-9 activity was assessed using “Caspase-9 Assay kit” K118 (Biovision, Milpitas, CA, USA). Cells were cultured in 6-well plates and treated with H2O2 (0.2 mM) in the presence or absence of Fucus spiralis VLC fractions (1 mg/mL) during 24 h. The cells were then washed twice with Hank’s buffer and collected by centrifugation at 5000 rpm during 10 min at 4 °C. The pellets were resuspended in 50 µL of lysis buffer and incubated on ice during 20 min. In order to separate the content of intracellular cytoplasmic organelles and cell membrane, centrifugation took place at 13,000 rpm during 20 min at 4 °C. Thereafter, 50 μL of supernatant was placed into a 96-well plate, to which was added 50 μL of reaction buffer containing DTT (10 mM) and 5 μL of substrate. This reaction was followed at wavelengths of 400 nm (excitation) and 505 nm (emission) along 90 min at room temperature. Caspase-9 activity was calculated by the slope of the fluorescence resulting from 7-amino-4-(trifluoromethyl) coumarin accumulation and expressed in % of control (Δ fluorescence (u.a)/mg of protein/min).
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