Gallios facs analyzer

Cells were counted using flow cytometry and Flow-Count fluorospheres (Beckman Coulter, Brea, CA, USA). After washing, harvested cells were resuspended in PBS containing 10 % FCS, 2 mM EDTA, and 7-aminoactinomycin D. Immediately prior to analysis, 50 μl of Flow-Count fluorospheres were added. Absolute cell counts were automatically determined using a Gallios FACS analyzer (Beckman Coulter, Brea, CA, USA). The system software calculated cell numbers using the following formula: cells per microliter = [(viable cells counted)/(fluorospheres counted)] × fluorospheres/microliter (see Additional file 1 for supplementary methods).

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).

Cells were harvested by treatment with 0.5 mM EDTA and resuspended into single cells. The cells were fixed in PBS with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were washed twice with cold PBS and incubated with methanol for 30 min for permeabilization. In the case of experiments involving the analysis of FGFRs on the cell surface, the permeabilization step was excluded. Then cells were blocked with PBS containing 0.5% BSA for 60 min at room temperature. The cells were washed and hybridized to the appropriate primary antibody at 4°C overnight. The cells were washed thrice with PBS and hybridized to the appropriate secondary antibody in PBS containing 0.5% BSA at 1:1,000 dilution for 1 h at room temperature. The cells were washed thrice with PBS and the fluorescence profiles were acquired in the Gallios FACS analyzer (Beckman Coulter). All the FACS data were analyzed using FlowJo software (BD Biosciences).