Ly6g pe

Forty-eight hours or 7 days after CLP procedure, animals were sacrificed by pentobarbital i.p. injection and blood and organs were harvested. Blood count was determined by a hemocytometer, and plasma concentrations of IL1β, IL-6, IL-10, IFNγ, and TNFα were measured by multiplex immunoassays (Bio-Plex Pro Mouse Th1 cytokine, Biorad, France) according to the manufacturer’s recommendations. The same protocol was carried out on healthy mice (H0).
Leukocyte trafficking was analyzed by flow cytometry as previously described [33 (link)]. The spleen and liver were crushed in HBSS and filtered on a 70-μm nylon filter. The bone marrow was extracted from the femur, after the bone has been clipped, by rapidly injecting 1 ml of PBS into the medullary cavity. The lungs were cut into fine pieces and incubated in a cocktail of collagenase I and DNase I at 37 °C for 45 min before being crushed and filtered. After washing, a cell count was performed by a hemocytometer with Trypan blue staining (BioRad). Cell suspensions were labeled with a combination of anti-CD4-PerCP, CD25-PE, CD11b-Vioblue, Ly6C-FITC, Ly6G-PE, FoxP3-APC, and CD45-PerCP mAbs (Miltenyi, France) after permeabilization according to the manufacturer’s recommendations. Data were acquired on a Gallios FACS analyzer (Beckman Coulter). The same protocol was carried out on healthy mice (H0).

Skin window chamber microscopy was done using a Nikon confocal AX microscopy (Nikon, NY, USA) at day 3 and day 7 post-skin window chamber implantation. 100 μL of antibody mixture per mouse: 0.15 µg Ly6G-PE (Miltenyi, CA, USA) and 0.15 µg CD8a-APC (Miltenyi, CA, USA) in PBS, was administered through retro-orbital injection. Microscopic imaging occurred 30 min post-antibody injection using 3 fields of view per mouse at 20X objective. KPCY6419 tumors were visualized within the skin window chamber using YFP. Images were analyzed using the Nikon Elements software (Nikon, NY, USA) using the automated measurement analysis macro. An individual threshold was applied to each imaging channel, and positive Ly6G-PE or CD8a-APC positive cells were quantified using the following analysis parameters; size: 5 µm to 10 µm, circularity: 0 to 1, smooth: 2X to 4X, fill holes: on and clean: 2X. The average of the 3 fields of view from each imaging time point for each animal was calculated.