Wallac microbeta counter

DNA synthesis was carried out as described in Ref. (7 (link)). The rate of proliferation of R⁻ IR-A cells was such that a sufficient difference between stimulated and unstimulated cells was not easily achieved. Therefore, the well-characterized L6 rat skeletal myoblasts stably overexpressing human IR-A were used. Briefly, L6 rat skeletal myoblasts (1.5 × 10⁴ cells/well), stably overexpressing human IR-A, were plated in a 96-well flat-bottom plate and grown overnight at 37°C, 5% CO₂. Cells were starved in SFM for 4 h before treatment with insulin, IGF-II, qIGF-I, or S597 with increasing ligand concentrations for 19 h in Dulbecco’s minimal essential medium with 1% bovine serum albumin. The cells were pulsed with 0.14 μCi/well [³H] thymidine for 4 h and harvested onto glass fiber filters (Millipore^®) using a MICRO 96™ Skatron harvester (Molecular Devices). The filters were counted in a Wallac MicroBeta counter (PerkinElmer Life Sciences).

BM-DC were cultured as previously described (Singh et al., 2009a (link)). BM of Myd88/Ticam1 DKO (referred to as MyD88/TRIF DKO) and Ctss^-/-(referred to as Cat-S KO) mice was kindly provided by Dr. T. Sparwasser (Twincore, Hannover, Germany) and Dr. K. Rock (Massachusetts Medical School, Worcester, MA, USA), respectively. CD11c⁺ spDCs were isolated as previously described (Singh et al., 2009a (link)). OVA-specific CD4⁺ and CD8⁺ T cells were isolated from spleen and lymph nodes cell suspensions from OT-II and OT-I mice, respectively, using the mouse CD4 and CD8 negative isolation kit (Invitrogen, CA, USA) according to manufacturer’s protocol. T cell proliferation assays were performed as described (Singh et al., 2009a (link)). In short,DCs were pulsed with OVA-Le^X or OVA for 4h before incubation with OVA-specific OT-I or OT-II T cells (2:1 DC:T). [³H]-Thymidine (1 µCi/well; Amersham Biosciences) was present during the last 16h of a 72h culture. [³H]-Thymidine incorporation was measured using a Wallac microbeta counter (Perkin-Elmer). Alternatively, OT-I or OT-II T cells were labeled with CFSE and after 3 days dilution of CFSE was analyzed by flow cytometry. Differentiation of naive OT-II T cells, induced by OVA-Le^X or OVA -pulsed BM-DCs or spDCs, was measured by an in vitro Th differentiation assay described earlier (Singh et al., 2011 (link)).

Isolated CD4⁺ T cells were stimulated and cultured in DMEM 1× (no glucose, no glutamine) with 5% NaHCO₃, 10% dialyzed FBS, 14 mM d-glucose or RPMI 1640 (Sigma–Aldrich) supplemented with 10% heat inactivated FBS (Sigma–Aldrich); 2 mM glutamine (Sigma–Aldrich) 0.5% Penicillin-Streptomycin (Sigma–Aldrich). 5 × 10⁶ T cells were incubated in 150 μl of media under hypoxia (O₂ = 1%) or normoxia (O₂ = 21%) for 72 h during which H³-thymidine (1 μCi/well; PerkinElmer, Boston, MA, U.S.A.) was added to the media for the last 12 h of incubation. H³-thymidine incorporation on the 72nd hour was counted using a Wallac MicroBeta Counter (PerkinElmer).