Tissue culture treated dishes

Experiments were performed on wild-type MEFs derived in the Brasier Laboratory^{40 (link)}. Line derivation was conducted under vertebrate animal protocols approved by the UTMB Animal Care and Utilization Committee. The cell line was routinely tested against mycoplasma contamination by DAPI staining and PCR. Cells were cultured in adherent conditions on tissue culture-treated dishes (Falcon) in complete Dulbecco’s modified medium (DMEM) with 4.5 g/l of D-glucose and 0.1 mM L-glutamine (ThermoFisher Scientific), with addition of 10% fetal bovine serum (ThermoFisher Scientific) and 100 mg/ml penicillin/streptomycin mix (Sigma-Aldrich). The culture was maintained in a conditioned incubator at 37 °C and 5% CO₂. For stimulation, cells were seeded on multi-well plates or coverslips and allowed to adhere overnight at 37 °C. Mouse recombinant TNF, LPS from Escherichia coli 0111:B4 (purified by ion-exchange chromatography) and CHX were purchased from Sigma-Aldrich. LPS was solubilized in a bath sonicator for 15 min and vortexed vigorously for additional 1 min prior to adding to the cells. CHX was added to the cells at a final concentration of 5 μg/ml 30 minutes prior to the addition of TNF or LPS.

Bone marrow cells or splenocytes were cultured in the presence of L929-cell conditioned medium as a source of colony stimulating factor [33 (link)]. The cells were cultured in RPMI1640 supplemented with 20% fetal bovine serum (Gibco, cat. 12657–029), 30% L929 cultured media, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. Cells were seeded in tissue culture treated dishes (BD Biosciences) and incubated at 37°C.
To culture spleen macrophages under the condition that is similar to those derived from bone marrow, we seeded 6X more spleen cells than bone marrow cells into culture dishes because the number of hematopoietic stem cells in the spleen was 6X lower than those in bone marrow. Despite this, the cultured bone marrow-derived macrophages became confluent at 5 days whereas spleen-derived macrophages required 12 days to reach the same degree of confluency.