Human neutrophils possess the remarkable ability to deform in a rapid, controlled manner during processes such as migration through tissue and phagocytosis of pathogens. However, the fundamental mechanisms behind neutrophil motility remain incompletely understood; for instance, what drives cell deformation during phagocytosis - passive adhesive attraction or active cytoskeletal protrusion? We addressed this question by testing how changes in IgG density affected the rate of neutrophil phagocytic spreading over flat surfaces. In this case, a glass coverslip coated with IgG acted as a model pathogen surface, eliciting a strong spreading response from neutrophils. We tested spreading on four different densities of IgG, ranging from tens of molecules per square micron to tens of thousands of molecules per square micron. We imaged spreading cells using reflection interference contrast microscopy (RICM), yielding high-contrast images of cell-substrate contact regions from which we could reliably quantify contact area growth over time. Remarkably, our data showed that the speed of spreading was essentially identical on the different IgG densities tested, and the maximum contact area only increased slightly as a function of IgG density. We concluded that phagocytic spreading is not passively driven by adhesion, but requires active protrusive stress exerted by the cell. This conclusion was confirmed by leveraging our data against a computational model of frustrated phagocytosis in our paper, "Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells". Further information on these experiments can be found in a companion experimental paper, "Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces".

Glass coverslips were coated with bovine serum albumin (BSA), followed by anti-BSA IgG antibody. Effective IgG density was varied by using different ratios of polyclonal rabbit anti-BSA IgG (strongly recognized by human neutrophils) to monoclonal mouse anti-BSA IgG-1 (not strongly recognized by human neutrophils. For example, for the 1% surface, the ratio of rabbit to mouse IgG was 1:99. The total concentration of IgG during this incubation was the same in all cases (150 µg/mL). The absolute IgG concentration on each surface was quantified by labeling IgG-coated surfaces with a fluorescent secondary antibody against rabbit IgG and comparing the brightness to a suitable standard (see companion manuscript for more details).

Human neutrophils were isolated from whole blood via immunomagnetic negative selection and were then deposited onto IgG-coated coverslips and imaged using reflection interference contrast microscopy (RICM).

For additional information on these methods, please refer to our companion manuscript, "Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces".

The data is contained in either ".txt" or ".dat" files, which can be opened using common text editors. They are formatted to be easily uploaded into spreadsheet programs such as Microsoft Excel. Please refer to the README file for details on the formatting of the attached data files, and contact Emmet Francis at emmet.a.francis@gmail.com with any further questions or concerns.