Figure 6.

Effects of mesenchymal stem cells (MSC) conditioned medium (CM) on neutrophil extracellular trap (NET) formation. A, Neutrophils were incubated with MSC CM or medium, then incubated with live S. aureus for 30 minutes or 2 hours, as noted in the Materials and Methods section, then fixed and immunostained for detection and quantitation of NET formation, using confocal microscopy. Total NET area was normalized to DAPI cell count, and was digitized and quantitated using ImageJ software, as described in the Materials and Methods section. Bars depict the total area at 30 minutes (black) or 2 hours (gray) following exposure to S. aureus. *** denotes P < .0005 as assessed by ANOVA and Tukey multiple means post‐test. Each experiment was conducted using MSC CM obtained from three different donor MSC, and neutrophils were collected from three unrelated healthy donors. B, Representative ×10 magnification images of NET formation by neutrophils 30 minutes (top row) or 2 hours (bottom row) after exposure to S. aureus. Red, green, and blue depict histone H3, neutrophil elastase, and DAPI expression, respectively. The upper right corner of each image depicts shows NET total area, calculated by Image J software, with colors inverted for clarity. C, Representative ×40 magnification images of neutrophil NETs, imaged under same conditions as described for (B)