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.
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.
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