Culture and differentiation of THP-1 cells and phagocytic assay.

All tissue culture media and reagents were obtained from Gibco. THP-1 cells (ATCC TIB-202) were grown in 95% air–5% CO₂ at 37°C in complete RPMI medium (RPMI with phenol red, supplemented with 10% fetal calf serum, 10 mM HEPES, 50 IU/ml penicillin, and 50 µg/ml streptomycin). Ten-centimeter dishes were seeded with 3.5 × 10⁶ THP-1 cells and differentiated by adding phorbol 12-myristate 13-acetate to a final concentration of 10 ng/ml and incubating them for 3 days at 37°C in 95% air–5% CO₂. Differentiated cells attach to the plastic surface. Following the 3 days of incubation, cells were washed twice with complete RPMI and then rested at 37°C in 95% air–5% CO₂ in complete RPMI for a further 3 days. At 90% confluence, differentiated THP-1 cells were incubated with bacteria (100 µl) at a 10:1 ratio of bacteria to THP1 cells. After 45 min, plates were washed and cells were reincubated with 10 μg/ml penicillin and 200 μg/ml gentamicin (Sigma, Germany) for 30 min to kill extracellular pneumococci. Intracellular bacteria were enumerated after lysis with 0.01% Triton X-100 by plating serial dilutions on blood agar.