Islets isolated from wild-type and Oxtr−/− mice were isolated and cultured in RPMI medium supplemented with 5% FBS overnight. Subsequently, only size-matched and well-shaped islets were transferred to a 12-well plate (20 islets/well), with each well represents one replicate. The islets were treated with 100 pM OXT (Sigma), 1 nM AVP (Sigma), 2 ng/mL Tunicamycin (Sigmga-Aldrich), or a cytokine mixture [IL1-β (50 U/mL) •TNF-α (1 × 103 U/mL) •INF-α (1 × 103 U/mL), Wako] for 24 hours. Cell death was measured by the Cell Death Detection ELISA Assay Kit (Roche) according to the manufacturer’s protocol. The cell death level was measured as the absorbance at 405 nm with respect to a substrate solution blank. The experiment was repeated for three times. The n number represents the total number of replicates from these independent experiments.
Cell death detection elisa assay kit
Manufactured by Roche
The Cell Death Detection ELISA assay kit is a laboratory equipment product that provides a quantitative method for the detection of cytoplasmic histone-associated DNA fragments, which are indicators of apoptosis or cell death.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using cell death detection elisa assay kit
1
Islet Cell Death Assay
2
Apoptosis Analysis in TGF-β Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells plated in 6-well plates were incubated with or without 4 ng/ml TGF-β in 0.2% FBS medium for 24 h. Then the cells were harvested and washed in cold PBS. Assay was performed using FITC Annexin V Apoptosis Detection kit (BD Biosciences, San Jose, CA) according to manufacturer’s instructions. Each sample was stained with FITC-Annexin (5.0 μl) and propidium iodide (5.0 μl), and percentage of apoptotic cells were evaluated after analysis using FACSCAN flow cytometer (Becton-Dickinson FACS Canto II). Apoptosis assay was also performed with the Cell Death Detection ELISA assay kit (Roche), which quantitatively determine DNA fragmentation using both anti-histone and peroxidase-conjugated anti-DNA antibodies. Whenever indicated, cyclophosphamide (CTX) (Sigma-Aldrich, St Louis, MO, C0768) and/or Afatinib (Santa Cruz Biotechnology, Dallas, TX, CAS439081–18-2) were added 30 min before TGF-β stimulation. For each treatment, triplicate experiments were performed.
3
Proton Pump Inhibitor Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
These
assays were performed as previously described.5 (link),40 (link),41 (link) Briefly, cells were seeded in six-well plates
(100 cells/well for PANC-1 and 200 cells/well for BxPC-3) and cultured
for 24 h before addition of PPIs or DMSO vehicle. The cells were continuously
cultured in the presence of PPIs or DMSO for 10–14 days followed
by staining with crystal violet and counting manually.
For the
apoptosis assay, BxPC-3 cells were seeded in 12-well plates (18 000
cells/well) and cultured for 24 h before treatment with lansoprazole
or DMSO control for 72 h. The cells were then harvested and subjected
to analysis using the cell death detection ELISA assay kit (Roche)
according to the manufacturer’s instructions. For detection
of cleaved PARP, BxPC-3 cells were seeded in six-well plates (200 000
cells/well) and cultured for 24 h before treatment with lansoprazole
or DMSO control for 24 h. Cells were then collected and subjected
to Western blot analysis of cleaved PARP using an antibody specific
to the cleaved PARP (Cell Signaling).
assays were performed as previously described.5 (link),40 (link),41 (link) Briefly, cells were seeded in six-well plates
(100 cells/well for PANC-1 and 200 cells/well for BxPC-3) and cultured
for 24 h before addition of PPIs or DMSO vehicle. The cells were continuously
cultured in the presence of PPIs or DMSO for 10–14 days followed
by staining with crystal violet and counting manually.
For the
apoptosis assay, BxPC-3 cells were seeded in 12-well plates (18 000
cells/well) and cultured for 24 h before treatment with lansoprazole
or DMSO control for 72 h. The cells were then harvested and subjected
to analysis using the cell death detection ELISA assay kit (Roche)
according to the manufacturer’s instructions. For detection
of cleaved PARP, BxPC-3 cells were seeded in six-well plates (200 000
cells/well) and cultured for 24 h before treatment with lansoprazole
or DMSO control for 24 h. Cells were then collected and subjected
to Western blot analysis of cleaved PARP using an antibody specific
to the cleaved PARP (Cell Signaling).
4
Apoptosis Analysis in TGF-β Treated Cells
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!