Cell Death Detection ELISAPLUS

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- When the absorbance of samples too high,reduce agent-concentration and decrease incubation period. - When the absorbance of samples too low, increase the agent concentration and prolong the agent incubation interval

Downstream tips
- When the absorbance of samples too high,reduce agent-concentration and decrease incubation period. - When the absorbance of samples too low, increase the agent concentration and prolong the agent incubation interval

Publication protocol

For determining apoptosis in THP-1 cells, apoptotic cells were quantified using a cell death detection ELISAplus kit (Roche Molecular Biochemicals, Mannheim, Germany). Cells (1×104) were incubated with 5, 10 and 25 nM of triptolide for 48 h. Cells were lysed with cell lysis buffer (200 μl). Cell lysates were assayed for DNA fragments using the cell death ELISAplus kit according to the manufacturer’s protocol. DNA fragmentation was measured at 405 nm against an untreated control. To measure the enzymatic activity of caspase proteases, a caspase colorimetric assay kit (R&D Systems, Inc. Minneapolis, MN, USA) was used. THP-1 cells (2×106) were treated with 5, 10 and 25 nM of triptolide for 48 h. Cells were harvested and cell pellets were lysed in 50 μl of lysis buffer on ice for 10 min. The proteins concentration in the supernatant (cytosolic extract) was measured by BCA assay. The activities of caspase-3-, -8 and -9-like proteases were measured by proteolytic cleavage of substrates, including DEVD-pNA (caspase-3 substrate), IETD-pNA (caspase-8 substrate) and LEHD-pNA (caspase-9 substrate) respectively. These colorimetry substrates were solubilized in an assay buffer. After incubation with the substrates at 37°C for 1 h in the dark, color production in the lysates was measured with a microplate reader at 405 nm. Caspase-3, -8 and -9 activities were determined by direct comparison to the level of the uninduced control. To assess the effect of caspase inhibitor treatment, THP-1 cells (1×104 cells) were pretreated with a pan-caspase inhibitor, Z-VAD-FMK, or a caspase-3-specific inhibitor, Z-DEVD-FMK (R&D Systems), for 2 h, followed by addition of 50 nM triptolide. After 48 h, cell viability was determined by a colorimetric assay with PMS/MTS solution. The absorbance was determined at 492 nm with background subtraction at 650 nm

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Apoptosis assay cell type - THP-1 using Cell Death Detection ELISAPLUS from Sigma-Aldrich.

Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Cell Death Detection ELISAPLUS below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Apoptosis assay cell type - THP-1 using Cell Death Detection ELISAPLUS from Sigma-Aldrich. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.