Generation and Culture of Mouse Embryonic Fibroblasts

MEFs were generated by standard methods from wild type, STING-deficient or cGAS-deficient mice. Detailed laboratory protocols are available upon request. All mice were on the C57Bl/6 background and obtained from both genders. Tmem173^-/- (STING-deficient) mice were a gift from J Cambier 49 (link). Mb21d1^-/- (cGAS knockout first) mice were described in 50 (link). Ifnar1^-/- were a gift from C Reis e Sousa and were originally described in 51 (link). This work was performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines for animal care. This work was approved by a project license granted by the UK Home Office (PPL No. 40/3583) and was also approved by the Institutional Animal Ethics Committee Review Board at the University of Oxford. Cells were cultured under 5% CO₂ and 5% O₂ at 37°C in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies) containing 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin (100 IU/ml)/streptomycin (100 μg/ml), 4.5 g/l D-glucose and 2 mM L-Glutamine. In some experiments MEFs were incubated at ambient O₂ or 40% O₂. Cells were repeatedly tested for mycoplasma using specific primers. WI-38 cells were purchased from ATCC CCL-75™ and cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) penicillin (100 IU/ml)/streptomycin (100 μg/ml). No method of cell line authentication was used.