Rat anti mouse ly6g

Single cell suspensions were prepared from peritoneal lavage samples or in vitro cell preparations. Cell counts were determined using a Coulter AcT 10 cell counter (Beckman Coulter, Brea, CA). Cells were suspended in FACS buffer (PBS with 1% bovine albumin and 0.1% azide) and nonspecific binding to cells was prevented by adding 5% rat serum (Invitrogen, Life Technologies, Grand Island, NY) and 1ul/sample of Fc Block (BD Pharmingen, San Jose, CA). These cells were stained with the following antibodies: Rat Anti-mouse Ly6G (BD Bioscience, Clone: 1A8, San Jose, CA), F4/80 (BD Bioscience), and FITC Annexin V (BD Bioscience). Neutrophils were characterized by expression of Ly6G, and peritoneal macrophages by expression of F4/80. FACS analsysis was conducted and analyzed using expression was determined using an Attune Acoustic Focusing Cytometer (Attune, Life Technologies, Grand Island, NY).
Neutrophil parent cell identification of microparticles was conducted using rat Anti-mouse Ly6 labeling. Microparticle populations were sized on forward- and side-scatter gates using 0.5-, 0.9-, and 3.0-μm latex calibration beads as previously described (38 (link)).

It has previously been shown that vessel dilatation and neutrophil accumulation reached a peak at 12 to 18 hours after corneal epithelial injury (23 (link)). Thus, after 18 hours from debridement surgery, mice were euthanized, and the eyes were enucleated. Tissues (n>3 per group) were fixed in 4% paraformaldehyde for 1 hour and the globes were dissected in phosphate-buffered saline (PBS) under a dissecting microscope, leaving the corneas with complete limbi. After washing three times with PBS, corneas were blocked in 2% bovine serum albumin for 15 minutes and permeabilized with 0.15 Triton X-100/2% bovine serum albumin for 1 hour. The corneas were then incubated with the following antibodies; anti-Ki67(Invitrogen 42569880, Carlsband, CA), rat anti-mouse Ly6G (BD Biosciences 565369, San Jose, CA), and hamster anti-mouse γδTCR (Invitrogen 17571181). At least three replicate corneal samples were imaged for each marker. Z stack corneal images were analyzed using ImageJ (Version 1.51, https://imagej.nih.gov/ij/) and subsequent manual counting of positively stained cells confirmed accuracy. Immunofluorescent cells at central, paracentral, peripheral and limbal regions (each presumed to occupy ¼ of corneal radius) of the cornea were counted. Wound edge is defined as occupying two adjacent 20x microscopic fields from the ridge of proliferating epithelial cells.