Bi-fluorescent infection enables single-cell analysis of intracellular killing .

Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of by neutrophils can be monitored using a strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent killing assay for use Conjugating with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed , and cells that have viable internalized . We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the utility of ATTO647N/GFP bi-fluorescent , we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.