Lymph nodes and minced tumors were digested using a cocktail of 1.67 Wünsch U/mL Liberase TL (Roche) and 0.2 mg/mL DNAse (Roche) as previously described [40 (link),64 (link)]. After lysis of red blood cells single cell suspensions were filtered through a 40-μm nylon cell strainer. Live cells were distinguished using Fixable Viability Dye eFluor 660 (eBioscience) prior to flow staining. All samples were incubated with anti-mouse CD16/32 (Fc block) for 10 minutes followed by staining for various surface markers for 30 minutes at 4°C. The following antibodies were used (all purchased from eBiosciences): PE-Cy7-conjugated anti-mouse CD11c, Alexa Fluor 700-conjugated anti-mouse CD86, PE-conjugated anti-mouse CD80, PE-conjugated anti-mouse CD1d, PE-conjugated anti-mouse CD70, FITC-conjugated anti-mouse CD40 and Alexa Fluor 700-conjugated anti-mouse MHC Class I (H-2Kd/Dd), FITC-conjugated anti-mouse CD27, PECy7-conjugated anti-mouse CD3ε, APC-conjugated anti-mouse CD122, Alexa Fluor 700-conjugated anti-mouse CD4. iNKT cells were identified by staining with mouse CD1d/PBS-57 or CD1d/unloaded tetramers (NIH Tetramer Core Facility) for 30 minutes at room temperature [65 (link)]. All samples were analyzed using a BD LSR II flow cytometer and FlowJo version 8.7.5 (Tree Star).
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