One million cells per well were seeded in 6-well plates. After 24 h, the cells were treated with 20 μM A4 for 3, 6 and 24 h. The cells were collected with phosphate-buffered saline (PBS) and washed again with PBS. The cells were centrifuged at 500 × g for 10 min. Cleaved caspase-9 activity was then measured in these samples by Caspase-9 Fluorometric Assay Kit (by BioVision cat#K118) according to the manufacture’s protocol. Fluorescence levels were measured by an ELISA spectrophotometer (400 nm excitation and 505 nm emission). The results were normalized by the absolute value (OD) of the DMSO control to evaluate the caspase-9 activity.
Caspase 9 fluorometric assay kit
Manufactured by Abcam
Sourced in United States
The Caspase-9 Fluorometric Assay Kit is a laboratory equipment product designed to detect and measure the activity of caspase-9, a critical enzyme involved in the apoptosis (programmed cell death) pathway. The kit provides a fluorometric-based method for quantifying caspase-9 activity in cell lysates or purified enzyme preparations.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3 fluorometric assay kit, by Abcam (2 mentions)
Adp atp ratio assay kit, by Abcam (1 mentions)
Annexin 5 cy3 apoptosis detection kit plus, by Abcam (1 mentions)
Mitoprobe transition pore assay kit, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Caspase 12 fluorometric assay kit, by Abcam (1 mentions)
Muse oxidative stress kit, by Merck Group (1 mentions)
Muse mitopotential assay kit, by Merck Group (1 mentions)
Muse multicaspase kit, by Merck Group (1 mentions)
F 4010, by Hitachi (1 mentions)
4 protocols using caspase 9 fluorometric assay kit
1
Caspase-9 Activity Measurement
2
Mitochondrial Dysfunction and Apoptosis Assays
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MitoProbe Transition Pore Assay Kit (M34153, Molecular Probes), TMRE (Molecular Probes), ADP/ATP ratio assay kit (ab65313, Abcam), Annexin V-Cy3 Apoptosis Detection Kit Plus (K202-100, Biovision), Caspase-9 Fluorometric Assay Kit (K118-100, Biovision) and In Situ Cell Death Detection Kit (11684795910, Roche) were used for mPTP, ΔΨm, ATP/ADP ratio and apoptosis assay, respectively. A total of 20 images representative of each group were chosen at random, and mean fluorescence intensity was analyzed by Leica Physiology Software (Leica). The experiments were repeated six times in triplicate, and a mean was calculated. For Cyt-C release, mitochondrial and cytosol fractions were separated by mitochondria Isolation Kit (89874, Pierce, Rockford, IL, USA) and the Cyt-C was detected by western blot. The experiment was repeated three times.
3
Apoptosis and Oxidative Stress Profiling
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Total caspase activation was detected by a Muse Multicaspase Kit (Merck Millipore, Billerica, MA, USA), and individual caspase activation was measured by a Caspase-3 Fluorometric Assay Kit, Caspase-8 Fluorometric Assay Kit, Caspase-9 Fluorometric Assay Kit and Caspase-12 Fluorometric Assay Kit (BioVision) using 1 × 106 cells for each assay according to the protocol. Mitochondrial membrane potential was measured by a Muse® Mitopotential Assay Kit (Merck). The cell cycle was determined using a Muse Cell Cycle Kit according to the protocol. Furthermore, oxidative stress was detected by a Muse Oxidative Stress Kit (Merck).
4
Quantifying Apoptotic Caspase Activity
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Caspase-9 and caspase-3 enzyme activities were measured by proteolytic cleavage of the fluorogenic substrate LEHD-AFC and DEVD-AFC using the Caspase-9 Fluorometric Assay Kit and Caspase-3 Fluorometric Assay Kit (BioVision Inc., Milpitas, CA, USA). HL-60 cells were treated with fluvastatin or simvastatin for 48 h (control cells were incubated without statins). The cells were collected, washed in PBS, and lysed in lysis buffer provided by the kit. For the assay, a solution of cell lysates containing 50 μM substrate was incubated at 37°C for 1 h. The release of AFC from the substrate was measured fluorimetrically using a fluorescence spectrophotometer (F-4010; Hitachi, Tokyo, Japan) with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The results were corrected for protein content of the lysates and are expressed as the change in proteolytic cleavage of the substrate (pM) for 1 h/mg protein. The protein content of the cell lysate was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA).
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