In vitro interactions of bacteria with human MCs (HMC-1). (A) Double immunofluorescence staining for determination of extracellular/intracellular location of S pyogenes associated with MCs (bar, 3.5 μm). Extracellular bacteria were stained with polyclonal rabbit anti–S pyogenes antibodies, followed by Alexa green–conjugated goat antirabbit antibodies (Sigma-Aldrich). After several washes, the cells were permeabilized by 0.025% Triton X-100 in PBS and washed again, and intracellular (as well as extracellular) bacteria were stained by anti–S pyogenes antibodies, followed by Alexa red–conjugated goat antirabbit antibodies (Sigma-Aldrich). Exclusively extracellular bacteria are labeled in green (i), extracellular plus intracellular bacteria are labeled in red (ii), and MC nuclei are labeled in blue (iii). An overlaid merged image where extracellular bacteria are labeled yellow and intracellular bacteria in red is shown (iv). (B) Transmission electron microscopic examination of cross-sections of HMC-1 cells cocultured with S pyogenes (bar, 2.5 μm). Bacteria are indicated by black arrows. Notice that all streptococcal microorganisms are extracellularly located. (C) Immunofluorescence staining for determination of extracellular (yellow) and intracellular (red) location of S aureus associated with MCs (bar, 3.5 μm). (D) Transmissionelectron microscopic examination of cross-sections of HMC-1 cells showing internalized S aureus (i,ii; white arrows). Extracellular bacteria (i) are indicated by a black arrow (bars, 1 μm). (E) Growth of S pyogenes in medium alone, in coculture with MCs, in coculture with cytochalasin D–treated MCs, or in coculture with MCs separated by a transwell system. Data are expressed as x-fold increase in bacterial growth with respect to the original inoculum. Each point represents the mean plus or minus SD of 3 independent experiments. *P < .05 by F-test for S pyogenes growth in medium control versus S pyogenes growth in the presence of either untreated or cytochalasin D–treated HMC-1 cells.