Spleen dissociation medium

Spleens were collected from mice of all three groups, the uninfected control, E. hellem-infected and LPS treated. Isolation of DCs starts with mince the spleen into homogenous paste using commercially available Spleen Dissociation Medium (STEMCELL Technologies, Vancouver, Canada) in tissue culture dish, followed by gently passing several times through a 16 Gauge Blunt-End Needle attached to a 3 cc Syringe and then through a primed 70 μm nylon mesh filter. The filtered single cell suspension was then subjected to the EasySep Mouse Pan-DC Enrichment Kit (STEMCELL Technologies, Vancouver, Canada) to isolate dendritic cells only. This kit targets non-DCs for removal with biotinylated antibodies recognizing specific cell surface marker on unwanted cells in solution. Briefly, Enrichment Cocktail and Biotin Selection Cocktail, combinations of monoclonal antibodies, were added to the sample. Next, the samples were incubated with Streptavidin-bound magnetic particles to solution and a magnet was used to immunomagnetic negative selection of the dendritic cells. The purified DCs were then counted and cultured in RPMI Medium 1640 (supplemented with 10% FBS, 50 ng/mL GM-CFS (granulocyte-macrophage colony-stimulating factor) and penicillin/streptomycin) (Gibco, Waltham, MA, USA) in a 37 °C, 5% CO₂ incubator.

Primary cancer and adjacent normal tissues were obtained from consenting patients undergoing surgery with the approval of the University of Alberta Health Research Ethics Board. Samples were received in saline and processed within 2 h of surgery. Submucosal and necrotic tissues were stripped from the tumor tissue and the remaining tumor was processed in 4 ml of “spleen dissociation medium” (STEMCELL Technologies) using a GentleMACS dissociator (Miltenyi Biotec). After rocking at 37°C for 30 min, the suspension was reprocessed, EDTA was added to 10 mM, and the cells were incubated at room temperature for 10 min. The cells were centrifuged for 5 min at 350 g, washed, and plated in EpiLife medium (Thermo Fisher Scientific) supplemented with 25 μg/ml bovine pituitary extract, 0.5 ng/ml epidermal growth factor, 3 mM glycine, 0.1 mM MEM non‐essential amino acids, 1% ITS (insulin, transferrin, selenium), 2% FBS, 2 mM l‐glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 μg/ml Fungizone^® (Gibco).

Lung tissue was digested with spleen dissociation medium (Stemcell), and mechanically dissociated using a GentleMacs Dissociator (Miltenyi). Cells were then strained through a 70 μm filter and washed with Hanks’ Balanced Salt Solution supplemented with 10% foetal bovine serum (FBS) followed by red blood cell lysis. For adoptive transfer experiments, penicillin–streptomycin (Gibco) was added to media throughout the experiment.

Spleens were harvested from 6 weeks-old C57BL/6 female mice dissected into 1 mm-sized portions and placed in commercially purchased Spleen Dissociation Medium (STEMCELL Technologies) for 30 min at 37°C and 5% CO₂. Then the splenocytes were passed through a 70 μm filter, centrifuged at 200 × g for 5 min, and resuspended in HBSS +2% HI-FBS + 1 mM EDTA.