Hmw polyinosinic polycytidylic acid poly 1 c

TruCulture^® tubes (Myriad RBM, Inc., Austin, TX, USA) were purchased and loaded with 10⁴ particles of heat-killed Bifidobacterium breve (Bb15700), Fusobacterium nucleatum (Fn23726), Fusobacterium nucleatum (Fn25586), or 1 ug/mL high molecular weight (HMW) polyinosinic:polycytidylic acid (Poly I:C) [all strains from ATCC, Manassas, VA, USA; HMW Poly I:C from InvivoGen, San Diego, CA, USA]. TruCulture^® tubes that were not loaded with HK bacteria served as a negative control. Human Whole-blood was drawn into TruCulture^® tubes as per manufacturer instructions, tubes were inverted 3-5X, and incubated in a dry heat block for 24 h (+/− 15 min) at 37 ℃ without agitation. N = 3 independent donors.

HEK293T cells (CH3 BioSystems) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies) in a 5% CO₂ incubator. HMW polyinosinic-polycytidylic acid (poly(I:C)) (InvivoGen) and polydeoxyadenylic-deoxythymidylic acid sodium salt (poly(dA:dT)) (InvivoGen) were prepared in accordance with the manufacturer's protocol and mixed with Lipofectamine 2000 (Life Technologist) at a ratio of 1:1 (μg/μl) in Opti-MEM (Life Technologies) for cell stimulation. The following antibodies were used: anti-DDX41 (Abcam), anti-Flag (Sigma), and anti-Myc (Sigma).