Ti 2 epifluorescent microscope

Immunostaining was performed as described previously⁸⁰ . Briefly, 1×10⁶ cells in 200 μL PBS were applied to a coverslip pre-coated with 10% poly-L-lysine. Cells were fixed with 4% methanol-free formaldehyde for 30 minutes at 37°C, permeabilized with 0.2% Triton-X 100 for 10 minutes at room temperature, and blocked with 1% BSA for 1 hour. Cells were incubated with a 1:500 dilution of anti-Zld or anti-Grh primary antibody overnight at 4°C. After washing with PBS, cells were incubated with a 1:500 dilution of goat anti-rabbit IgG DyLight 488 conjugated secondary antibody (Thermo Fisher Scientific #35552) for 1 hour at RT. Imaging was performed on a Nikon Ti2 epi-fluorescent microscope with a 60× objective lens.

Briefly, polyacrylamide gel substrates were polymerized on a 25 millimeters (mm) glass coverslip, using 3% acrylamide and 0.2% bisacrylamide for a Young’s modulus of E = 1.5 kPa, along with fluorescent red 0.5 $\mu \mathrm{m}$ carboxylate-modified polystyrene beads. Gel substrates were then coated with human fibronectin (Gibco 33016015) using the photoactivatable crosslinker sulfo-SANPAH (Sigma 803332) and rinsed extensively. Further experimental details are documented elsewhere in Oakes et al.^{73 (link)} and Witt et al.^{60 (link)}, respectively. The polyacrylamide gel and coverslip were mounted in a coverslip holder, then covered with 1 mL of Leibovitz L-15 media. About 50,000 neutrophils were plated and allowed to adhere for 15 minutes. Approximately 20-60 adherent cells were imaged in phase microscopy using a Nikon TI-2 epifluorescent microscope using a 40X air objective with a 0.6 numerical aperture. An Okolab enclosure around the TI-2 maintained the apparatus at 37° and 5% CO₂ for the duration of the experiments. Only adherent cells were selected for imaging. The $N$ represents the number of individual neutrophils imaged and analyzed, with an $n>3$ for individual septic or healthy donors.