Figure 4: Enucleated cytoplasts formed after NETosis are intact and retain functional responses.

Neutrophils (PMN) and neutrophil cytoplasts were flow sorted from HDM/LPS treated mouse lungs (protocol day 3). (A,B) The morphology and size of the sorted PMN and cytoplasts were determined by phase contrast microscopy. ****p < 0.0001 by two-tailed Student’s t-test. (C-E) Chemotaxis to LTB4 was assessed using a microfluidics device with sorted cells (see Methods) (C) Fluorescent microscopic image showing one chemotaxis unit in the microfluidic device. (D) PMN or (E) cytoplasts were loaded into the microfluidics chamber with an LTB4 (100nm) gradient and time lapse, phase-contrast microscopic imaging was performed for 6 hours (Representative images). (F) Measurements of the number of cells that entered the migration microchannels per unit at various conditions. ****p < 0.0001 using a one-way ANOVA. (G) Measurements of chemotaxis velocity of PMN in the migration channels and chemokinesis velocity of cytoplasts in the cell loading channel ****p < 0.0001 using a one-way ANOVA. (H) Individual trajectories of cytoplast chemokinesis in the cell-loading channel. (I-K) Phagocytosis by PMN and cytoplasts was determined using pHRodo-coupled E. coli particles. (I) The absence of nuclei in cytoplasts confirmed by DAPI staining. (J) Phagocytosis of pHrodo-E.coli particles leading to fluorescent color change in PMN and cytoplasts. (K) Phagocytosis index (%Total) was determined. This experiment was performed 3 times. Values represent the mean and error bars SEM. (L) The killing capacity of sorted neutrophils and cytoplasts towards Streptococcus pneumoniae (serotype 1) was determined at different time points indicated. This experiment was performed 2 times. Values represent the mean between duplicate controls and error bars SEM used in a representative experiment.