Apoxin green indicator and 1 7 aad apoptosis necrosis detection kit

A549 lung epithelial cells were grown to confluency on fibronectin-coated circular glass coverslips in 24-well tissue culture plates and then incubated with 50 μg/ml (2.9 μM) mucoricin or 5 μg/ml (77 nM) ricin (concentrations shown to cause in vitro damage to alveolar epithelial cells [Fig. 3f]) for 2 hours after which the cells were washed and stained with 1x Apoxin Green Indicator and 1× 7-AAD (Apoptosis/Necrosis detection kit, Abcam) for 45 min. The cells were fixed and mounted in ProLong Gold antifade containing DAPI (Life Technologies) to visualize cells. Microscopic z-stack pictures were taken using a Leica SP8confocal laser scanning platform. Apoptotic cells vs. necrotic cells were identified by their green and red fluorescence, respectively. The number of apoptotic and necrotic events per high-power field (HPF) was determined, counting 10 HPF per coverslip. The experiment was performed three times in triplicate.