Mycobacterial preparation for macrophage infections

Bacteria for infection were prepared as described previously (7, 22). Briefly, Mtb were grown to late-log phase, and washed repeatedly with PBS. After the final wash, bacterial clumps were removed by slow-speed centrifugation (300×g), and the resulting supernatant was then sonicated to break up any remaining clumps and generate a single-cell suspension. After sonication, the bacterial OD600 was recorded and the bacteria were diluted into macrophage-infection media (RPMI plus 10% horse serum [THP1 and U937 cells] or 10% autologous human serum [primary cells]).