Cd8a bb515

Tumors were minced and digested by using the mouse Tumor Dissociation Kit (Miltenyi Biotech), washed with FACS buffer (PBS containing 2% FBS, 2 mM EDTA and 0.02% sodium azide), and filtered through a 70 -µm filter (BD Biosciences). The obtained single-cell suspension was stained with LIVE/DEAD Fixable Blue Dead Cell Stain kit (Thermo Fisher Scientific), blocked with anti-CD16/32 (BD Biosciences; #553142; clone 2.4G2; 1:1000), stained with fluorochrome-labeled antibodies, and analyzed using a LSR Fortessa (BD Biosciences) and FlowJo software (Tree Star Inc).
The following antibodies were purchased from BD Biosciences: CD3-BUV737 (#564380; clone 17A2; 1:400), CD8a-BB515 (#564422; clone 53-6.7; 1:400), CD44-APC-Cy7 (#560568; clone IM7; 1:1000), CD274-BV711 (#563369; clone MIH5; 1:200), CD11b-FITC (#557672; clone M1/70; 1:800), CD45-BV605 (#563053; clone 30F11; 1:400). The following antibodies were purchased from Biolegend: CD4-BV510 (#100449; clone GK1.5; 1:400), CD62L-BV785 (#104440; clone MEL-14; 1:200), CD161-PE (#108707; clone PK136; 1:400), CD11c-PE (#117308; clone N418; 1:200), F4/80-AF647 (#123122; clone BM8; 1:2000).

Blood samples were collected from tail vein of 2 months old mice (n = 7 L and n = 7 LnL) into EDTA coated tubes. Whole blood was treated with Lysis buffer (BD) to eliminate erythrocytes before adding fluorochrome-labeled antibodies to bind specifically to leukocyte surface antigens. Cells were analyzed by flow cytometry (BD FACS CAntoII, BD Biosciences Becton Drive Franklin Lakes, NJ, USA) using combinations of the following monoclonal antibodies: CD45 BV510 (563891 BD), CD4 PECY7 (552775 BD), CD8a BB515 (584422 BD), CD11b APC (553312 BD), CD3 BB700 (566494 BD), CD19 PE (557399 BD). 10,000 events were acquired and stored for each analysis.
Lymphocytes were identified by their characteristic appearance on a dot plot of FSC versus SSC. Fluorescence overlap was then compensated electronically using lymphocytes stained with single colors. Data were analyzed using FlowJo after singlet doublet discrimination and live cells (Fixable viability stain 700 BD bioscience). Leukocytes were identified with SSC and CD45 gates. All populations were identified as subpopulation of CD45 positive cells and reported as percentage of leukocytes.

To assess and quantify inflammatory cells infiltration, lungs of ADAM28^+/+ and ADAM28^-/- were harvested and digested in collagenase C (1mg/ml; Gibco) prior to red blood cell lysis (Red Blood Cell Lysis Buffer, Sigma Aldrich, Saint-Louis, Missouri). Cells were stained with fluorochrome-conjugated surface antibodies during 30 minutes, fixed and permeabilized using Cytofix/Cytoperm (ref#554714 BD Biosciences, Erembodegem, Belgium) before intracellular antigen staining. Antibodies used for flow cytometry analysis were: CD5-BV421 (ref#53-7.3, BioLegend), CD4-PERCP CY 5.5 (ref#RM4-5, BD Biosciences), CD8a-BB515 (ref#53-6.7, BD Biosciences), IFN-γ-PE CY7 (ref#XMG 1.2, BD Biosciences), IL-4-APC (ref#11B11, eBioscience, Thermo Fisher Scientfic), CD45R-APC-eFluor780 (B220) (ref#RA3-6B2, eBioscience). Data were acquired on FACS CANTO II flow cytometer (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences).