Salmonella minnesota lps

Raw 264.7 murine macrophages were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in tissue culture flasks. Raw cells were harvested via trypsinization and subcultured at 5 × 10⁵ cells per well in 24-well plates for 24 h to allow attachment overnight before experimentation.
For BMM preparation, femurs and tibias were removed from euthanized female CD1 mice and flushed with sterile PBS through a 70 µm filter. The cells were centrifuged for 5 min at 4 °C and 500× g and resuspended in 28 mL complete DMEM (cDMEM) (10% FBS, 5% horse serum, 1% HEPES, 1% sodium pyruvate, 1% GlutaMAX, 1% penicillin–streptomycin). Then, L929 conditioned media (7 mL) containing murine CSF was prepared and added to the cDMEM, as described previously [21 (link)]. Macrophage differentiation was carried out for 12 days before experimentation.
Macrophages (Raw 264.7 or BMMs) were then treated with purified LT, purified ST, Salmonella minnesota LPS (InvivoGen, cat# R595, San Diego, CA, USA), IL-33 (R&D Systems, cat# 3626-ML, Minneapolis, MN, USA), forskolin (Sigma, cat#F3917, Waltham, MA, USA), CFA/I (BEI Repository, cat# NR-49110, Manassas, VA, USA), or deferoxamine mesylate (Sigma, cat# D9533).

Cells were plated in flat-bottomed 24-well plates at a density of 2.5 x 10⁶ cells (for RBC-lysed total bone marrow cells or total splenocytes) in 500 μl of complete cell medium, or in flat-bottomed 96-well plates at a density of 50,000 cells (for HSPC-derived macrophages) in 200 µl of complete cell culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin stock solution [Gibco]). Whole blood was diluted 1:2 in complete cell culture medium at a final volume of 200 μl and plated in flat-bottomed 96-well plates. To prevent potential fungal growth, 0.5 μg/ml amphotericin B was added to the cultures of total bone marrow cells, total splenocytes and blood from infected animals. Cells were challenged with 100 ng/ml of Pam₃CSK₄, 100 ng/ml of ultrapure Salmonella minnesota LPS (all from In vivogen) or 25 x 10⁶ inactivated C. albicans ATCC 26555 yeasts for 24 h, and cell-free supernatants were then harvested and tested for cytokine release using commercial enzyme-linked immunosorbent assay (ELISA) kits [TNF-α and IL-6 (eBioscience)]. Unstimulated cells served as negative controls. Triplicate samples were analyzed in each assay.