File Name : figure s1.tif

Caption : figure s1. chemotaxis of isolated human neutrophils to a chemoattractant gradient. 
a) loading of microchambers followed by washing of the outer chamber creates a gradient along the connecting channel, here visualized using fluorescein. in this example, isolated human neutrophils loaded into the outer chamber chemotax up a gradient of fmlp into the inner microchamber. 
b) measurement of fluorescence (left y-axis) in the inner (light green) and outer (dark green) chambers over time indicates the evolving chemokine gradient generated in this device. recruitment of isolated human neutrophils (blue, right y-axis) begins to plateau towards 7-8 hours as the difference in fmlp concentration between the two chambers diminishes. 


File Name : figure s2.tif

Caption : figure s2: visualizing loss of neutrophil viability and netosis.
a) frames extracted from time lapse imaging of neutrophil recruitment to ltb4 in the presence (lower panels) and absence of pma (upper panels). pma induced netosis from 3-4 hours, as indicated by diffuse pi staining (open yellow arrows). scale bar: 100 µm.
b) graphing of viable neutrophil counts and pi stain area over 8 hours of imaging. ltb4 alone (left graph) induced robust neutrophil recruitment with low pi staining. the presence of pma (right graph) resulted in reduced neutrophil recruitment and viability due to strong, consistent induction of netosis. 
c) frames extracted from time lapse imaging comparing neutrophil interactions with s. aureus in which the bacteria prevails (upper panels) to interactions in which neutrophils prevail (bacteria co-loaded with ltb4, lower panels). proliferation of bacteria in the lower panels is associated with increased loss of neutrophil viability (bright pi staining, solid yellow arrowheads) and induction of netosis (diffuse pi staining, open yellow arrowheads). scale bar: 100 µm.
d) graphing of neutrophil counts compared to bacterial (gfp) and pi fluorescent signal shows that onset of exponential bacterial growth correlates with loss of neutrophil viability and netosis,  as indicated by a drop in viable neutrophil count and increase in pi fluorescence. 
statistics: n = 9 assays scored per condition, error bars: mean  sem.


File Name : figure s3.tif

Caption : figure s3. detailed statistical analysis of neutrophil recruitment to s. aureus compared to fmlp (fig 6a).
a) recruitment of neutrophils from three healthy donors (black, blue and red) in response to s. aureus. significant inter-donor differences are indicated for each condition. statistics: one-way anova with multiple comparisons test. *p<0.05, ***p<0.001, ****p<0.0001. each point represents a microchamber.
b) recruitment of neutrophils from three healthy donors (black, blue and red) in response to fmlp. significant inter-donor differences are indicated for each condition. statistics: one-way anova with multiple comparisons test. ***p<0.001, ****p<0.0001. each point represents a microchamber.
c) comparison of intra-sample variation between responses to s. aureus and fmlp using coefficient of variance. recruitment to s. aureus was significantly more variable than fmlp between assays of samples from the same donor. statistics: two-tailed t-test.  ****p<0.0001.