Tlr3 agonist poly 1 c

Peripheral blood was collected via an intravenous arm draw at 1 year of age and again at 4 years of age (see Supplemental Table 2 for a timeline of Behavioral assays and blood draws). 4 mLs of peripheral blood was collected into acid-citrate-dextrose Vacutainers. Blood was centrifuged at 2100 rpm for 10 mins and plasma was collected and aliquoted into 2 mL cyrovials and stored in −80 °C until cytokine analysis was performed. The remaining blood cells were layered on lymphocyte separation medium (Corning; Manassas, VA) and PBMC were separated by gradient centrifugation. PBMC were washed twice with 50 mLs each with Hanks Balanced Salt Solution (Corning; Manassas, VA). Cells were counted, excluding dead cells based on trypan blue staining, and PBMC concentrations were adjusted to a concentration of 1 × 10⁶ cells/mL in complete media (RPMI 1640 (Invitrogen; Carlsbad, CA) with 10% Fetal Bovine Serum (Corning; Manassas, VA), 100 IU/ml penicillin (Invitrogen; Carlsbad, CA) and 100 IU/ml streptomycin (Invitrogen; Carlsbad, CA), 1% L-glutamine (Invitrogen; Carlsbad, CA)). Cells were stimulated with TLR4 agonist (LPS; 50 mg/mL) (Sigma-Aldrich; St. Louis, MO) or TLR3 agonist (Poly I:C; 100 mg/mL) (Sigma-Aldrich; St. Louis, MO). After 48 hours, supernatants were collected and stored at −80 °C until analysis.