Hemacolor

The lower sides of the Transwell inserts with 5.0 μm pores (Corning) were coated with 10 μg/cm² MFAP4 or HSA overnight at 4°C, washed with PBS, blocked with 10 mg/ml HSA for 1 h at room temperature and washed again. The monocytes were seeded in the upper chamber (100.000 cells/insert) in serum-free RPMI medium containing 0.5% FBS. In some experiments, the cells were pre-incubated with anti-integrin α_Vβ₃, anti-integrin α_Vβ₅ (both from Merck Millipore), or isotype control antibody (Thermo Fisher) for 30 min at room temperature before seeding. The lower chamber contained serum-free RPMI medium with 0.5% FBS ± 100 ng/ml human recombinant CCL-2 (R&D Systems). The cells were allowed to migrate for 3 h, after which the upper sides of the filters were washed with PBS and swiped with a cotton swab to remove any non-migrated cells. The lower sides of the filters were then stained with Hemacolor (Sigma) and divided into four fields. The migrated cells in each field were counted in a blinded manner by two independent investigators.

Blood smears were prepared from ∼10 μl of peripheral whole blood taken from a subset of alligators (Supplementary Table S2). Blood smears were prepared in the field and allowed to air dry at room temperature on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA, United States). Slides were stained with Hemacolor® according to the manufacturer’s protocols (Sigma-Aldrich, St. Louis, MO, United States; Cat: 1.11661). Peripheral whole blood cell counts were made by evaluation of stained blood smears using 40X and 100X objectives on a Nikon Eclipse 80i microscope (Nikon; Melville, NY, United States). Each slide was examined independently by three different investigators blinded to sampling site, size, and sex. Leukocyte identity was determined by cellular morphology and staining characteristics (Stacy et al., 2011 (link); Sykes and Klaphake 2015 (link)). A minimum of 400 leukocytes and thrombocytes were counted to calculate percentage of each cell type. Leukocytes were categorized into lymphocytes, basophils, heterophils, eosinophils, azurophils, and basophils with percent of each cell type determined by dividing the number of each cell type by the total cells counted minus the number of thrombocytes and then multiplying by 100. Variation between individual researcher counts was determined to be less than 7.9% for each cell type.

Bronchoalveolar lavage (BAL) was performed on lungs from terminally anesthetized mice by washing with 400 μL of ice-cold phosphate-buffered saline (PBS) followed by a further three washes of 300 μL. Cells were pelleted, BAL fluid (BALF) was taken from the first lavage, and then all lavages were pooled. Cell counts were determined by hemocytometer and are presented as total cells of recovered volume which ranged between 1.03 mL and 1.21 mL. Cells were centrifuged onto glass slides and stained using Hemacolor® (Sigma-Aldrich, Darmstadt, Germany).