In vitro assay of macrophage interaction with nontypeable Haemophilus influenzae (NTHi). Bone marrow–derived macrophages from C57BL/6 and myeloid differentiation primary response gene 88 (MyD88)−/− mice were incubated with NTHi for 1 h, after which they were treated with a high concentration of gentamicin that killed all extracellular bacteria. Recovery of viable internalized bacteria from cell lysates was then assessed after an additional 1, 2, or 5 h of culture with gentamicin (n = 6 wells per time point). Results are shown as box plots encompassing the interquartile range; horizontal lines represent median values, dots represent mean nos. of colony-forming units per well, and error bars represent extremes of the data. After 1 h of gentamicin treatment, macrophages from C57BL/6 mice contained significantly more NTHi than did macrophages from MyD88−/− mice (P < .02). After 2 h of gentamicin treatment, the no. of colony-forming units recovered from wild-type (WT) macrophages had declined dramatically (P < .01), whereas recovery from MyD88−/− macrophages was unchanged (P = .18). By 5 h, most bacteria recovered from WT and MyD88−/− macrophages were nonviable. Comparing the 1- and 2-h time points, C57BL/6 macrophages killed significantly more internalized NTHi (P < .03) than did MyD88−/− macrophages (P = .70).