Calpain has been implicated in the apparent expansion of neutrophil plasma membrane that accompanies cell spreading and phagocytosis. In order to test this hypothesis, an internally quenched fluorescent peptide substrate of calpain-1 which increased in fluorescence on cleavage, was micro-electroinjected into neutrophils. The fluorescence intensity increased in a significant number of neutrophils, including those which appeared to be in a morphologically resting (spherical) state. In order to test whether calpain was activated by an elevation of cytosolic Ca2+ during the injection, Ca2+ chelators were added to the injectate and cytosolic free Ca2+ in the receiving neutrophil was simultaneously monitored. It was shown that this approach could be used without raising Ca2+ within the injected cell. Despite this, approximately 75% of individual neutrophils had calpain activity which consumed the substrate within approx. 100 s. It was found that all neutrophils had elevated calpain activity were phagocytically competent; whereas neutrophils with low or undetectable calpain activity failed to undergo phagocytosis. This association was consistent with the hypothesis that calpain activity within neutrophils was necessary for them to undergo efficient phagocytosis. (C) 2015 Elsevier Inc. All rights reserved.