Intramacrophage survival assay

The U937 human monocyte cell line was obtained from ATCC. The intra-macrophage survival assay for S. Typhi was performed as previously described [36] with slight modifications. The cells were grown in RPMI 1640 (Gibco, NY) supplemented with 5% fetal bovine serum (FBS; Gibco) and 50 U/ml penicillin-streptomycin (Life technologies, NY) at 37°C with 5% CO₂. Cells were centrifuged at 58 X g for 10 min, resuspended in fresh 37°C pre-warmed RPMI 1640 medium + FBS without antibiotics, adjusted to a concentration of ~1x10⁶ cells per ml, and 10 ml of cells were added to a 50 ml tube. After 24 hours, bacterial cultures were centrifuged and the bacterial pellets were resuspended in RPMI 1640 + FBS. The bacteria were added to U937 cells at a ratio of ~100 bacteria per U937 cell (time 0). After incubation at 37°C for 90 minutes, the infected U937 cells were centrifuged as before, washed 2 x with phosphate buffered saline (PBS) and resuspended in RPMI 1640 medium + FBS containing 250 μg/ ml of gentamicin in order to kill extracellular bacteria. After another 90 minutes (total time 3 hours), the cells were resuspended in 20 ml of fresh RPMI medium containing 25 μg/ml of gentamicin to prevent extracellular growth of any released bacteria. Additional PBS-washing of the cells was done at 24 hours post-infection. For viable bacterial count determinations at time 0, 3 and 24 hours, the infected macrophages were lysed with 0.1% Triton X-100 in PBS. After 3 min, cell lysates were serially diluted 10-fold in PBS, and aliquots were plated onto TSA plates to assess bacterial CFU. The bacterial CFUs were normalized by calculating bacteria per macrophage. All assays were repeated at least three times on different days. The results are presented as the mean ± SEM of all replicates. Statistical analyses were performed by means of an unpaired t test using GraphPad Prism version 5. A p value of <0.05 (two tailed) was considered to be statistically significant (**).