Dynamic Measurement Of The Membrane Potential Of Phagocytosing Neutrophils By Confocal Microscopy And SEER (Shifted Excitation And Emission Ratioing) Of di-8-ANEPPS

Abstract

Neutrophils engulf and kill invading microorganisms in a process called phagocytosis. Accompanying phagocytosis is the Respiratory Burst, a surge of activity by the enzyme NADPH oxidase. The enzyme transports electrons from the cytosol to the phagosome, where it creats ROS that help to kill bacteria. This electrogenic transport of electrons must be compensated for, so that the enzyme can function continuously. Here we report for the first time the membrane potential of individual phagocytosing neutrophils using a highly sensitive confocal techinque.Isolated human neutrophils were incubated with 10 μM di-8-ANEPPS for 15 minutes and allowed to adhere to glass coverslips. The cells were then washed with Ringer's solution and treated with either 60 nM PMA or opsonized zymozan (OPZ). SEER consisted of ratioing two simultaneously acquired images of fluorescence, excited at 488 nm and 545 nm and collected at emission ranges 470-560 nm and 570-700 nm respectively. Using this approach we were able to detect a 26 % change in ratio from −50 to +50 mV.Neutrophils stimulated with 60 nM PMA depolarized 80 ± 6 mV (n=5) after a delay of ∼ 3 minutes. Addition of the NADPH oxidase inhibitor DPI repolarized the membrane, confirming that the depolarization was due to NADPH oxidase activity. Neutrophils challenged with 2 mg/ml OPZ showed a depolarization spike of 86 ± 11 mV (n=5) that coincided with the onset of phagocytosis. In both cases the depolarization lasted several minutes before subsiding spontaneously.This study confirms that phagocytosis by human neutrophils is accompanied by a large depolarization of their plasma membrane, a depolarization that can now be monitored quantitatively and dynamically in individual cells.Suppport NIH-HLBI (HL61437), NIAMS (AR049184) & Philip Morris.