CD73 Monoclonal Antibody (AD2), PE, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Lungs were perfused with 10 ml PBS. A single-cell suspension was obtained by using C tubes (Miltenyi Biotec), followed by incubation with 1 mg/ml of collagenase D (Roche) and 0.1 mg/ml of DNase (AplliChem) for 1 h at 37°C. Cells were washed and suspended in PBS containing 3% FBS. Erythrocytes were lysed in ammonium-chloride-potassium lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, H2O), and cells were washed with PBS.	Finally, cells were incubated with Fc-Block antibody (anti-CD16/CD32; BD Biosciences) for 15 min at 4°C	Cells were analyzed with a MACSQuant cytometer system (Miltenyi Biotec) and with FlowJo software (Treestar).

Upstream tips
Lungs were perfused with 10 ml PBS. A single-cell suspension was obtained by using C tubes (Miltenyi Biotec), followed by incubation with 1 mg/ml of collagenase D (Roche) and 0.1 mg/ml of DNase (AplliChem) for 1 h at 37°C. Cells were washed and suspended in PBS containing 3% FBS. Erythrocytes were lysed in ammonium-chloride-potassium lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, H2O), and cells were washed with PBS.
Protocol tips
Finally, cells were incubated with Fc-Block antibody (anti-CD16/CD32; BD Biosciences) for 15 min at 4°C
Downstream tips
Cells were analyzed with a MACSQuant cytometer system (Miltenyi Biotec) and with FlowJo software (Treestar).

Publication protocol

Lungs were perfused with 10 ml PBS. A single-cell suspension was obtained by using C tubes (Miltenyi Biotec), followed by incubation with 1 mg/ml of collagenase D (Roche) and 0.1 mg/ml of DNase (AplliChem) for 1 h at 37°C. Cells were washed and suspended in PBS containing 3% FBS. Erythrocytes were lysed in ammonium-chloride-potassium lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, H2O), and cells were washed with PBS. Finally, cells were incubated with Fc-Block antibody (anti-CD16/CD32; BD Biosciences) for 15 min at 4°C and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (MAbs) specific for CD8, CD11b, F4/80, and B220. Allophycocyanin (APC)-conjugated MAbs specific for CD3 and Ly6G; phycoerythrin (PE)-conjugated MAbs specific for CD4, CD11c, and CD73; and a PE-cyanine 7-conjugated MAb specific for CD45.2 were also used. All the MAbs were purchased from eBioscience. Cells were analyzed with a MACSQuant cytometer system (Miltenyi Biotec) and with FlowJo software (Treestar).

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