Naio4

Cell-surface sialic acids were labeled using aniline-catalyzed oxime ligation (41 (link)). In comparison to labeling with Sambucus nigra lectin (SNA) (Vector Laboratories), which are large and have defined preferences for specific Sia linkages, chemical labeling provides a more reproducible and quantitative measurement of cell-surface sialic acids (Fig. S5). To perform this reaction, we first prepared solution A (1 mM NaIO₄ [Sigma-Aldrich] dissolved in PBS supplemented with 1 mM CaCl₂) and solution B (1 mM CaCl₂, 10 mM aniline [Sigma-Aldrich], 5% FBS and 100 μM CF633 hydrazide [Sigma-Aldrich], or CF488A hydrazide [Sigma-Aldrich] in cold PBS, pH 6.5 [Teknova]). The cells were cooled on ice and washed with cold PBS once before incubating with solution A on ice for 15 min, followed by a wash with cold PBS (pH 6.5) and incubation with solution B on ice for 40 min. The cells with labeled sialic acids were washed once with cold Opti-MEM, and their plasma membranes were labeled with CellMask Orange (Invitrogen) at a concentration of 2.5 μg/mL in Opti-MEM for 5 min at room temperature. The cells were washed again with Opti-MEM before imaging.

HA-CA conjugate was synthesized by modifying HA (MW 200 kDa, Lifecore Biomedical, Chaska, MN, USA) with a catechol group using dopamine hydrochloride (Sigma, St. Louis, MO, USA)^{34 (link),35 (link)}. In brief, HA was dissolved in distilled water at a concentration of 1% (w/v). Equal molar quantities of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (TCI Co., Tokyo, Japan) and N-hydroxysulfosuccinimide (NHS) (Sigma) (relative to the HA backbone unit) were then added into the solution and stirred for 30 min. dopamine hydrochloride was added to the solution at an equal molar ratio to HA, and the pH of the solution was adjusted to 5.0 using 1 M hydrochloride. The reaction was continued overnight by stirring at room temperature. To eliminate unreacted dopamine hydrochloride, the solution was dialyzed against 1× phosphate buffered saline (PBS) (3 M Korea, Seoul, Korea) at a pH of 5.0 four times each for 6 h and against distilled water once for 4 h. The resultant solution was then frozen and lyophilized. For gelation, the HA-CA pre-gel solution (dissolved in PBS) was mixed with a sodium periodate solution (NaIO₄, Sigma) at an equal molar ratio to catechol to oxidize the catechol groups of HA-CA to reactive o-quinones, which forms covalent crosslinks^{34 (link),35 (link)}. The pH condition of HA-CA hydrogel was adjusted to 7.2–7.4 using NaOH-containing NaIO₄ solution for cell culture.

Alginate (medium viscosity), Pluronic F-127, NaIO_4, NaOH, PBS, were purchased from Sigma-Aldrich. CTL was obtained from Proteogenix (France). A 1 cm diameter biomedical Ti₆Al₄V bar was procured from ACNIS International Society (France). The bar was cut in several discs (1 cm diameter/1 cm thick), then polished using silicon carbide papers from ESCIL (France) with a decreasing grain-size (600, 800, 1200, 1600, 2000, 2400, 4000).

SI was induced by injection of the chemical irritant sodium periodate (NaIO₄; 1ml of 5mM, i.p.; Sigma, Oakville, ON, Canada) in phosphate buffered saline (PBS) [12 (link)]. The mice were sacrificed 3h after peritoneal injection by CO₂ asphyxiation and peritoneal exudate was collected by lavage with 10 mL chilled PBS. Following 3 hours of NaIO₄-induced peritonitis, cells isolated from the peritoneum and mounted onto slides using Cytospin™ 4 Cytocentrifuge (Thermo Fisher Scientific). Greater than 90% of all cells were confirmed to be neutrophils by hematological stain (Diff-Quik, Siemens, Deerfield, IL). Neutrophils were counted by a hemocytometer (Bright-line, Hausser Scientific, Horsham, PA). Four counts were conducted per mouse, averaged, and expressed as a mean ± SEM per strain.