Ammonium chloride potassium lysis buffer

BMDCs were generated in accordance with a modified version of a method described previously (Helft et al., 2015 (link); Lou et al., 2004 (link)). Briefly, bone marrow cells isolated from the femurs and tibiae of 7–14-wk-old WT, Aim2^−/−, Sting^−/−, Cxcl10^−/−, Aim2^−/−Sting^−/−, Aim2^−/−Ifnar^−/−, Aim2^−/−Cxcl10^−/−, Il1β^−/−, and Il-18^−/− mice were filtered through a 70-µm nylon strainer, and red blood cells were lysed by ammonium-chloride-potassium lysis buffer (Sigma Aldrich) and cultured in BMDC medium (RPMI-1640 containing 10% FBS, 100 U/ml PS, 2 mM L-glutamine [Gibco], 50 µM 2-mercaptoethanol [Sigma Aldrich], 20 ng/ml GM-CSF [PeproTech], and 10 ng/ml IL-4 [PeproTech]).
The BMDC medium was replaced on days 3 and 6. On day 8, nonadherent cells were harvested, washed two times with PBS, and used for in vitro experiments. DC purity was assessed by flow cytometry to ensure staining for markers CD11c, MHC II, CD11b, and CD86 on BMDCs. For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO₂ with 10 µM of the human gp100_25–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.