The murine bone marrow was collected from femurs in RPMI (Life Technologies) + 10% FBS (R10 Omega Scientific) + 5% antibacterial/antimycotic (Life Technologies), and red blood cells were lysed using ACK lysis buffer. Bone-marrow-derived macrophages (BMDMs) were differentiated using MCSF (10 ng/ml; R&D Systems) and L929-conditioned media for 7 days. Non-adherent BMDMs were re-plated and treated the following day (see Supplemental Experimental Procedures ).
For Luminex analysis, CD4+ CD62L− CD44+ E/M T cells were sorted from splenocytes and cultured on anti-CD3-coated plates (BD Pharmigen) + CD28 soluble antibody 2 µg/ml (BD Pharmigen) added to the media (RPMI + 10% FBS + 5% antibacterial/antimycotic). After 96 hr, the supernatants were measured for IFNγ, TNFα, IL-4, and IL-5 by Bio-Plex Pro Mouse Cytokine Th1/Th2 Assay (Bio-Rad no. M60-00003J7) on Luminex xPONENT system.
For Luminex analysis, CD4+ CD62L− CD44+ E/M T cells were sorted from splenocytes and cultured on anti-CD3-coated plates (BD Pharmigen) + CD28 soluble antibody 2 µg/ml (BD Pharmigen) added to the media (RPMI + 10% FBS + 5% antibacterial/antimycotic). After 96 hr, the supernatants were measured for IFNγ, TNFα, IL-4, and IL-5 by Bio-Plex Pro Mouse Cytokine Th1/Th2 Assay (Bio-Rad no. M60-00003J7) on Luminex xPONENT system.