Easysep mouse apc positive selection kit 2

Bone marrow cells were isolated by flushing marrow from tibias and femurs using a 27-gauge needle and PBS. Cells were cultured at 1.5 x 10⁶ cells/ml for 7 days in RPMI 1640 media (Wisent) supplemented with 10% FBS (Wisent), 50 μM β-mercaptoethanol, antibiotic/anti-mycotic (Wisent) and 100 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L) (Peprotech, catalog no. 250-31L). On day 7, 10 ng/ml of Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, catalog no. 315–03) was added to the culture. For the stimulation of FLT3L-BMDCs, 10⁴ live or an antigenic preparation (obtained by two cycles of freeze and thaw) of B16F10 or LLC cells per million of DCs were added. In some stimulations, DCs were segregated from live cancer cells with a 0.4 μm cell culture insert (Falcon). For transfer experiments, FLT3L-BMDCs were stimulated on day 7 with GM-CSF and on day 9 with live B16F10 cells and harvested on day 10. Before the injection, XCR1⁺ FLT3L-BMDCs were isolated using XCR1-APC (Biolegend) and EasySep™ Mouse APC Positive Selection Kit II (Stemcell).

For the isolation of lung EOS, male and female WT mice were exposed by intranasal (in) instillation to 50 μL of 1 mg/mL of house dust mite (HDM) Dermatophagoidus pteronyssinus freeze dried extract (Citeq Biologics, Groningen, The Netherlands) for 10 consecutive days and then euthanized at day 11. Upon euthanasia, mice were tracheotomized with an 18-gauge catheter, and a BAL was conducted by performing three separate injections/aspirations of 1 mL saline solution. In order to eliminate most macrophages, BAL cells were incubated for 2 h in RPMI +10% FBS in a flask, and non-adherent cells were collected. EOSs were enriched from non-adherent cells by negative selection using EasySep Mouse APC Positive Selection Kit II (Stemcell Technologies, Vancouver, BC, Canada) with Ly-6G-APC (BioLegend, San Diego, CA, USA), CD90.2-APC (BioLegend, San Diego, CA, USA), CD19-APC (BioLegend) and F4/80-APC (BioLegend) antibodies to remove neutrophils, T cells, B cells and macrophages, respectively. The purity of the obtained mEOS suspensions was assessed by flow cytometry and microscopy using DiffQuik coloration (HemaStain Set, Hampton, NH, USA).