Salmonella Infection in Macrophage Model

THP-1 cells were obtained from ATCC (American Type Culture Collection: TIB-202) and grown in suspension in RPMI +Glutamax supplemented with 10% (v/v) FBS in a humidified 37°C, 5% CO₂ incubator. All cell culture reagents were from Life Technologies, unless otherwise stated. Low passage (passage 15 or less) cells were plated in either 8-chamber Ibidi dishes (Ibidi) or 24-well tissue culture treated plates (Costar) in the presence of phorbol 12-myristiate-12 acetate (PMA, Sigma-Aldrich). For cells plated 5 days prior to infection, PMA was removed after 2 days of treatment. Primary human monocytes were isolated using negative selection (Dynabeads Untouched Human Monocyte Isolation Kit, Life Technologies) from apharesed whole blood from healthy donors and plated in 8-well Ibidi dishes 7 days prior to infection in RPMI supplemented with 5% (v/v) Male AB human serum (Sigma), 2 mM L-glutamine (Cellgro), 1 mM NaPyruvate (Cellgro), 1X MEM NEAA, 1 mM Hepes and 100 ng/mL macrophage colony-stimulating factor (MCSF) (Peprotech). Media was refreshed on day 3 and day 5. Salmonella enterica serovar Typhimurium SL1344 wild type [29 (link)] and constitutively expressing mCherry [30 (link),31 (link)] have been previously described.