Neutrophils actively swell to potentiate rapid migration

While the involvement of actin polymerization in membrane protrusion is well-established, we have a more limited understanding of the role of transmembrane water flow in cell motility. Here we investigate the role of water influx in neutrophil migration. These cells undergo directed movement to sites of injury and infection. Chemoattractant exposure increases cell volume and potentiates neutrophil migration, but the causal link between these processes is not known. Using a genome-wide CRISPR screen, we identify the regulators of the chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for rapid migration following chemoattractant stimulation. Our data demonstrate that cell swelling complements cytoskeletal inputs for chemoattractant-induced potentiation of migration., In these experiments, primary human neutrophils were imaged using Fluorescence Exclusion Microscopy (FxM). FxM is a highly accurate method for measuring cell volume that relies on cells excluding a dye in a microfluidic chip with a flat ceiling held up by pillars. The height of chamber is known and can be used to determine the volume displaced by the cells. The FxM image is taken with a low magnification objective (in this case a 20x 0.75NA Plan Apo) with a depth of field larger than the height of the chamber to capture all of the fluorescence in the chip. For all experiments in this dataset, the excluded dye is a 10,000 MW dextran conjugated to AlexaFluor-647 and the light source is a Sutter Lambda LS with a Xenon Arc lamp. Two other channels were taken for segmentation purposes including a nuclear channel (a Hoechst stain imaged with a BFP filter set) and a cytoplasmic channel (Calcein Red-Orange imaged with a RFP filter set) also taken in epifluorescence mode. After 15 minutes of ima..., , # Neutrophils actively swell to potentiate rapid migration

Primary neutrophils were isolated from the blood of volunteers and then *gently* injected into the microfluidic chips. Mechanical stimulation activates the cells so extreme care was taken to keep the cells quiescent and unactivated. The FxM chips were prepped such that they always had 3 lanes per chip and all lanes were imaged concurrently to minimize the impact of timing and, if possible, all lanes were included in the analysis. One lane was usually an untreated (aka WT) condition and the other two lanes were different drug conditions (see `fxm_dataset_info.csv` for details). If lanes were discarded, the reason is included in the `fxm_dataset_info.csv` in the `notes` section. The primary reason for not including a lane was due to incomplete bonding of the microfluidic chip or due to flow in the microfluidic chip. The chips lack a resistive element so any bubbles in the system would lead to very large flows and the cells coul...