Splenic Leukocyte Isolation and Stimulation

Whole splenic leukocytes were isolated using standard laboratory procedures described in detail previously^{9 (link),38 (link),40 (link),49 (link),60 (link),61 (link)}. Briefly, the spleens were dissociated by gently scraping through a steel screen, and the cell suspension was passed through a 70-μm cell strainer to remove tissue debris. To eliminate the unintended in vitro exposure to estrogens, care was taken to use only charcoal-stripped FBS and phenol red-deficient media. The splenic leukocytes were adjusted to 5×10⁶/ml in complete medium for seeding into cell culture plates for stimulation. Briefly, the cells were plated into 48-well cell culture plates (0.25 ml/well), and stimulated with Imiquimod (5 µg/ml) (IDT Inc., Skokie, IL, USA), ODN-2395 (0.5μM, synthesized by IDT Inc.), ODN Control (IDT Inc.), or complete media for the designated time by adding an equal volume of 2x concentration of stimulation medium. CD4⁺ and CD19⁺ cell were purified from whole splenic leukocytes using the Miltenyi Biotec “CD4⁺ (L3T4) MicroBeads, mouse,” and “CD19⁺ MicroBeads, mouse,” per the company’s published manual separation protocol (Miltenyi Biotec Inc., Auburn, CA, USA). Cell pellets were washed with cold PBS and stored at −80 °C.