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One hundred microliters of supernatants from control neutrophils treated with either TNF or IL-1β were plated in a 96-well plate. Ten microliters of neutrophil elastase substrate (Z-Ala-Ala-Ala-Ala)2Rh110 (Cayman Chemical) was added to each well, and the plate was incubated for 1.5 hours at 37°C. Fluorescence was measured in a BioTek Synergy H1 Hybrid Multi-Mode Reader (Agilent). Results are reported in RFU.
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