Cell death elisa

Briefly, 96-well clear-bottomed plates were coated with anti-histone antibody for 1 h at RT. ARPE-19 total cell lysates (5 μg/well) were diluted in the incubation buffer to a final volume of 100 μl/well. Samples in duplicate were equilibrated on the coated plates for 90 min at RT. To measure apoptosis-associated DNA fragmentation, we followed the manufacturer’s protocol for Cell Death ELISA (Roche Life Sciences).

A total of 5 × 10³ cells were incubated in 6-well cell low attachment plates (Corning) for 72 h under standard conditions. Cells were then harvested, and anoikis was assessed using an Annexin V-FITC/PI cell apoptosis kit (BD Biosciences), a cell death ELISA (DNA fragmentation, Roche), and a FACSAria flow cytometer (BD Biosciences), all according to the manufacturers' instructions. The group of cells cultured in normal plates was considered the control group.

An in-house ELISA was used to quantify MPO-DNA complexes. First, Plasma was collected from the whole blood of mice by centrifugation at 500 ×g for 20 min at 4 ℃; mouse cerebral cortex samples were homogenized in the lysis buffer (1:10 dilution) and centrifugated at 1500 rpm for 15 min at 4 ℃ to collect the supernatant. Briefly, after overnight coating with anti-MPO antibody (1:500, 0400–0002, Bio-Rad) at 4 ℃, 96-well plates were then blocked with 5% BSA for 2 h at room temperature. After washing three times (300 μL), 50 μL of plasma or 50 μL of tenfold dilution of the mouse brain homogenates was added into the wells with 80 μL of incubation buffer containing a peroxidase-labeled anti-DNA mAb (Cell Death ELISA, 11774425001, Roche, Basel, Switzerland). The plates were then incubated at room temperature for 2 h. After five washes, the plate was developed with 100 μL ABST substrate. The absorbance (OD) at 450 nm was measured after 30 min incubation in the dark. All samples for completion and analysis of ELISA were performed by two researchers blinded to experimental groups.

Apoptosis was detected in vehicle- or drug-treated MBR cells with a cell death ELISA (Roche Diagnostics, IN). Briefly, cells (200,000 per time point) were treated with vehicle or the indicated drug for 24 or 48 h, and cell lysates were used for the detection of oligonucleosomes using an antibody against histone.