Mouse plasmacytoid dendritic cell isolation kit 2

As described previously [23 (link)], for the isolation of pDCs and neutrophils from the spleen, a cell suspension was obtained and subjected to purification after mechanical disruption and RBC lysis. Cells were enriched from total splenic cells by using the mouse plasmacytoid dendritic cell isolation kit II and mouse neutrophil isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany).
Neutrophil supernatants were obtained by means of overnight culture of 1 × 10⁶ purified splenic neutrophil in 100 μl of complete medium without stimulus. Neutrophil supernatants were harvested and clarified by means of centrifugation at 4°C (10,000g for 5 min), and aliquots were stored at -80°C until further use. For pDC stimulation, 1 : 5 dilution of neutrophil supernatant was added to 5 × 10⁵ total splenic pDCs, followed by culture for 4 hours or overnight for mRNA and ELISA analysis of IFN-α production.

C57BL/6 wild type, TLR7^-/- and TLR9^-/- double-knockout mouse femurs were provided by B. Pulendran, Emory University. Mice were maintained in specific-pathogen-free conditions at the Emory Vaccine Center vivarium in accordance with all animal protocols reviewed and approved by the Institute Animal Care and Use Committee of Emory University. Bone marrow cells were isolated by flushing femurs with PBS supplemented with 2% heat inactivated FBS. BM cells were resuspended in Tris-ammonium chloride at room temperature for 1 minute to lyse RBC, washed, then cultured in RPMI 1640 Glutamax (Invitrogen) with 10%FBS, 1nM sodium pyruvate, 10mM HEPES buffer, 100 units/mL penicillin, 100ug/mL streptomycin, 2mM L-glutamine, 1% MEM nonessential amino acids. BM cells were cultured for 8 days at 1 × 10⁶ cells/ml in 24-well plates in culture medium supplemented with 200 ng/ml recombinant murine Flt-3 ligand (Peprotech) as previously described [48 ]. After 8 days pDCs were purified from BM cells using the mouse plasmacytoid dendritic cell isolation kit II (Miltenyi) with a purity ranging from 85 to 95% and cells were phenotyped by CD11c PerCP and PDCA1 APC (BD Pharmigen).