DOI: 10.25338/b8134b
title: Data from: Integrative experimental/computational approach establishes active cellular protrusion as the primary driving force of phagocytic spreading by immune cells

description: 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".
description: 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".
description: 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.