Apoptosis Assay by Flow Cytometry Analysis

Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) stains were used to determine the percentage of cells within the biofilm undergoing apoptosis and necrosis on the electrodes during the bioelectrochemical denitrification operation. An apoptosis assay was conducted using the protocol supplied by the manufacturer BioVision, Inc. The cells were gently removed from the electrodes for a brief time, re-suspended with 500 μL of 1× binding buffer, and then treated with 5 μL of Annexin V-FITC and 5 μL PI. Immediately after a 5-min incubation in the dark at room temperature, each sample was analyzed using a FACSCalibur™ flow cytometer with the supplied software as the instrument. The Annexin V-FITC binding (Ex = 488 nm; Em = 530 nm) was analyzed using a FITC signal detector, and PI staining was analyzed by a phycoerythrin emission signal detector. A cytogram analysis was performed using the FLOW software version 2.4.1; the unstained cells were classified as “live,” “Left Bottom,” or “Q1 area.” Meanwhile, cells stained with Annexin V only were classified as “early apoptotic,” “Left Top,” or “Q2 area”; cells stained by both Annexin V and PI were classified as “late apoptotic,” “Right Top,” or “Q3”; cells stained by PI only were classified as “dead,” “Right Bottom,” or “Q4” cells.