Treatment of the Monocyte-Derived Dendritic Cells (Mo-DCs) with Sialidase

Collect approximately 10 times 10 to the sixth human monocyte derived dendritic cells differentiated from the monocytes. From the 10 wells of the 24 well plate having 1.3 times 10 to the sixth cells per well, transfer them to a new sterile 15 milliliter conical tube. After centrifuging the cells at 300G for five to seven minutes at room temperature, using the normal break, discard the supernatant to remove dead cells and debris.

Add 10 milliliters of RPMI 1640 medium containing 11.1 millimolar glucose to the tube. After centrifuging at room temperature for four minutes at 300G, using the normal break, discard the supernatant again. Add two milliliters of RPMI 1640 medium to the cell pellet and divide one milliliter of the cell suspension into two new sterile micro tubes labeled as number one and number two.

To micro tube one, add 500 milli units of Sialidase from Clostridium perfringens. Incubate both tubes for 60 minutes at 37 degrees celsius. After incubation, transfer the cells from both micro tubes into corresponding new 15 milliliter conical tubes labeled as number one and number two.

Then add approximately four milliliters of complete RPMI 1640 medium containing 10%FBS to each conical tube. Centrifuge the tubes at room temperature for four minutes at 300G using the normal break and add five milliliters of complete RPMI 1640 medium to the pellet. Plate one milliliter cells per well.

The effectiveness of Sailidase treatment for reducing the sialic acid content down the surface of human monocyte derived dendritic cells was evaluated by flow cytometry and confocal microscopy. Sailidase treatment significantly decreased Maackia Amurensis lectin and Sambucus nigra lectin binding while increasing Peanut agglutinin lectin staining. The decrease in Sambucus nigra lectin staining after Sailidase treatment showed a significantly reduced Sambucus nigra lectin staining at the cell's surface.