Rpmi glutamax

HT-29 cells were cultured in T75 cm² tissue culture flasks (Corning) in RPMI GlutaMAX (Gibco) supplemented with 10% Foetal Bovine Serum (FBS Bovogen Biologicals) in 5% CO₂ at 37°C. Two days before infection HT-29 cells were seeded into 24 well tissue culture trays at a density of 2x10⁵ cells per well. The day before infection derivatives of EPEC were inoculated into LB broth and grown with shaking at 37°C overnight. On the day of infection, overnight cultures of EPEC were sub-cultured 1:75 in RPMI GlutaMAX (Gibco) and grown statically for 3 h at 37°C with 5% CO2. Where necessary, cells were induced with 1 mM IPTG (Sigma) for 30 min before infection. HT-29 cells were washed twice with PBS and infected with EPEC grown to an OD₆₀₀ of 0.06 for 3 h before being lysed for immunoblot analysis. Where required, after 3 hours of EPEC infection the HT-29 cells were treated for a further 2 hours with 100 μg/ml gentamycin to stop the infection, in combination with 20 ng/ml FasL before being lysed for immunoblot analysis.

For primary BMDM culture, cells were flushed from both femurs of young BALB/c mice and cultured for 6 days in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Germany) supplemented with 1% sodium pyruvate, 1% L-glutamine, 2-mercaptoethanol, 1% penicillin-streptomycin (Gibco BRL), 10% fetal bovine serum (FBS; HyClone) and 10% L-cell conditioned media. At day 3, fresh media was added and BMMs were used for the experiments on day 7.
The variants of RAW264.7 and THP-1 cells used in this study were cultured as per the suppliers protocol (InvivoGen) in DMEM and RPMI-GlutaMAX, respectively (Life Technologies) with 10% heat-inactivated FBS. Murine RAW264.7 derived wild-type and knockout (KO) macrophages such as RAW-Lucia^TMISG, RAW-Lucia^TMISG-KO-STING, RAW-Lucia^TMISG-KO-cGAS, RAW-Lucia^TMISG-KO-TREX-1, RAW-Lucia^TMISG-KO-IRF3, RAW-Lucia^TMISG-KO-TBK1, RAW-Lucia^TMISG-KO-IFI16, RAW-Lucia^TMISG-KO-RIG1 and human monocyte THP1-Dual™, THP1-Dual™ ISG-KO-STING cells were all commercially purchased from Invivogen and used for in vitro experiments. Plated macrophages were incubated overnight at 37 °C to adhere at 5% CO₂, and then stimulated accordingly. The viability of cells was >98%, as assessed by trypan blue dye exclusion technique.

Leukemia cell lines (HL-60, K-562) were cultured routinely in RPMI Glutamax (Life Technologies) supplemented with 10% FBS and 1% solution of 10,000 units/mL penicillin and 10,000 μg/mL streptomycin. Glucose or glutamine-deprived medium were obtained from DMEM powder (Sigma). In some experiments, cells were treated with 10 mM 2-deoxy-D-glucose (2-DG), 3 μg/mL Tunicamycin (TUN) and/or 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH); all these compounds were obtained from Sigma. L-[³H]-glutamine and L-[³H]-leucine were purchased from PerkinElmer.

Human type-2 alveolar basal epithelial cells (A549) (ATCC CCL-185) were maintained in growth medium (RPMI Glutamax (Life Technologies) supplemented with 5% v/v newborn calf serum (NCS) (Life Technologies), 1mM L-glutamine (L-glut) (Life Technologies) and 1 × Penicillin/streptomycin (Life Technologies)). HEp-2 cells (ATCC CCL-23) were maintained in Opti-MEM media (Life Technologies) supplemented with 10% v/v NCS, 1mM L-glut and 1 × Penicillin streptomycin.

Different types of human leukemia cell lines were used: promyelocytic leukemia cells (HL-60), acute monocytic leukemia cells (THP-1), myelogenous cells (KG1a), acute monocytic cells (MV4–11) and erythromyeloblastoid leukemia cells (K-562). These leukemia cells were routinely cultured in RPMI Glutamax (Life Technologies) supplemented with 10% FBS. The Ba/F3 cell line transfected with Bcr-Abl (a gift from Dr. K. Bhalla, MCG Cancer Center, Medical College of Georgia, Augusta, GA, USA) was cultured in RPMI1640 medium supplemented with 10% FBS and 1% non-essential amino acids solution (Life Technologies). For the experiments requiring medium adaptation, Dulbecco's Modified Eagle Medium (DMEM) powder (Sigma) was used to generate media containing glucose (10 mM) and/or glutamine (2 mM), and MEM (Sigma) was used to produce medium containing serine or not; dialyzed serum (10%) was used in these experiments.