T cells were seeded in 96-well plates, activated with anti-CD3/CD28-coated beads and treated for 1 or 5 days with PMT. Caspase 3, 7, and 8 activities were measured using the Caspase-3/7Glo and Caspase-8Glo Assay (Promega) in accordance to the manufacturer’s instructions. Luminescence was measured using the Plate CHAMELEONTMV microplate reader (Hidex).
Caspase 8glo assay
Manufactured by Promega
Sourced in United States
The Caspase-8Glo Assay is a luminescent-based assay designed to measure the activity of caspase-8, a key enzyme involved in the initiation of apoptosis. The assay provides a quick and sensitive method for detecting and quantifying caspase-8 activity in cell-based samples.
Automatically generated - may contain errors
Lab products found in correlation
Caspase glo 3 7, by Promega (1 mentions)
Plate chameleontmv, by Hidex (1 mentions)
Ecl luminol reagent, by GE Healthcare (1 mentions)
Flow cytometer analysis, by Beckman Coulter (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Annexin 5 pi, by Keygen Biotech (1 mentions)
Plate reader luminometer, by Promega (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Roche (1 mentions)
Complete mini, by Roche (1 mentions)
4 protocols using caspase 8glo assay
1
Caspase Activity Assay in T Cells
2
Caspase-8 Activation Assay in BMDMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess caspase-8 activation, BMDMs were infected with fliI- or flaA- for 8 hours, and the activity of caspase-8 was measured using the Caspase-8 Glo Assay (Promega) according to manufacturer´s recommendations. To evaluate caspase-8 activation by western blot analysis, 1 X 107 cells were infected at a MOI of 10 and lysed 8 hours after infection in RIPA buffer with protease inhibitor, as previously described. The lysates were suspended in 4X Laemmli buffer, boiled for 5 minutes, resolved by 15% SDS PAGE and transferred to 0.22-μm nitrocellulose membranes. The membranes were blocked in Tris-buffered saline (TBS) with 0.01% Tween-20 and 5% non-fat dry milk or for 1 hour. The mouse anti-caspase-8 (Enzo– 1G12) and anti-rabbit peroxidase-conjugated antibody (KPL; 1:3000) were diluted in blocking buffer for the incubations (overnight for anti-cleaved caspase-8 and 1 hour for the secondary antibody). ECL luminol reagent (GE Healthcare) was used for antibody detection.
3
Melatonin and 5-Fu Induce Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell apoptosis and decrease of mitochondrial transmembrane potential induced by melatonin or 5-Fu was determined by AnnexinV/PI (KeyGEN, Nanjing, China) and rhodamine (Beyotime, Shanghai, China) staining, respectively, followed by flow cytometer analysis (Beckman Coulter, California, USA) according to manufacturer's instructions. Also, caspase activity was measured by Caspase 3/7 Glo assay and Caspase 8 Glo assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. The intracellular level of ROS was detected according to a previous report.42 (link)
4
Measuring Caspase-8 Activity in Retinas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Attached and detached retinas were harvested after 3 days and homogenized using a sonicator at 20% power for 10 pulses in lysis buffer (20 mM MOPS, pH 7.0; 2 mM EGTA; 5 mM EDTA; 0.1% Triton X-100). 1 tablet of protease inhibitor (Complete Mini; Roche Diagnostics, Indianapolis, IN) and 1 tablet of phosphatase inhibitor (PhosSTOP; Roche Diagnostics, Indianapolis, IN) per 10 mL were added to the lysis buffer. The homogenates were centrifuged at 10,000 g for 10 minutes at 4 °C, and total protein concentration of the supernatant was determined using a Micro BCA protein assay kit (ThermoScientific, Rockford, IL). A luminescent assay kit (Caspase 8 Glo Assay, Promega, Madison, WI) was utilized to measure caspase 8 activity according to the manufacturer’s instructions. In short, 50 μg of protein was incubated with substrate in a white-walled 96 well-plate at room temperature for 1 hour. The attached retinas served as controls. Luminescence was measured in a plate reader luminometer (Turner Biosystems, Sunnyvale, CA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!