Splenocyte isolation and challenge with PMA and ionomycin.

Spleens were isolated from wild-type and EPX^-/- mice challenged via intraperitoneal inoculation with antigen, either 50 µg ovalbumin (Sigma) or 20 µg A. fumigatus extract (Hollister-Stier) complexed with Imject Alum (Pierce). Splenocytes were generated from spleens minced in Hanks Balanced Salt Solution with 1% fetal bovine serum and 10 mM HEPES and then passed through a 100 micron cell strainer. Splenocytes were pulled into a 10 ml syringe and passed through a 21-gauge needle, followed by centrifugation at 300 x g for 5 minutes. Cell pellet was resuspended in 1 ml of HBSS, 1% FBS, 10 mM HEPES and red blood cells were lysed with distilled water followed by 10X PBS. Cells were enumerated using trypan blue dye exclusion and resuspended at 5 x 10⁶ cells per ml of RPMI 1640 plus 10% fetal bovine serum (Atlanta Biologics {"type":"entrez-protein","attrs":{"text":"S11150","term_id":"98016","term_text":"pir||S11150"}}S11150), Penicillin 10 units/mL and Streptomycin 10 µg/mL (Life Technologies) and challenged with combined phorbol 12-myristate 13-acetate; (PMA; Sigma) at 20 ng/mL) and ionomycin (Sigma) at 1 µg/mL for 3 hours. IL-3 released into the supernatants was evaluated by ELISA using the Quantikine R & D kits as per manufacturer’s instructions.