Regulation of TLR9/10 expression by the mitogens anti-μ and anti-CD40. (A) Purified resting tonsillar B lymphocytes were stimulated in vitro for 24 hours and 48 hours (2 ×10⁹ cells/L) with or without anti-μ (10.5 μg/mL) + CD40 Ab (0.5 μg/mL). Total RNA was prepared from control resting B cells (R), buoyant in vivo–activated B cells (GC), and αμ/CD40-stimulated B cells.^24,48  (B) Purified resting tonsillar B lymphocytes were stimulated in vitro for the indicated time periods (2 × 10⁹ cells/L) with anti-μ (10.5 μg/mL), anti-CD40 Ab (0.5 μg/mL), or anti-μ in combination with CD40 Ab (0.5 μg/mL). Total RNA was prepared and cDNA synthesized and analyzed for expression of TLR9 and TLR10 by real-time PCR as outlined in “Materials and methods.” β-Actin expression was used to standardize all results. Results are expressed as copy number × 10⁶/μg RNA added to the cDNA synthesis reaction (mean ± SEM [n = 3] for a representative experiment). These results are representative of 3 independent experiments.