Bovine Dendritic Cell Growth Kit

1. Prepare peripheral blood mononuclear cells (PBMC) from heparinised blood by density gradient centrifugation.

2. Purify CD14^+ve cells by labelling PBMC with CD14 mAb and utilise magnetic bead or flow cytometric separation techniques.

3. Resuspend the isolated CD14^+ve cells at a concentration of 1x10⁶ cells/ml in tissue culture medium (TCM = RPMI or equivalent + 10% foetal calf serum) containing a final dilution of 1:20 of PBP015KZZ .

4. Add 3ml of cell suspension to each well of a 6 well tissue culture plate.

5. Culture cells in a humidified atmosphere of 5% CO₂ in air, at approximately 37^oC.

6. Culture cells for 3 days. The cells may then be harvested and used for other procedures including immunophenotyping (as required).

7. If a longer culture period is required the cells must be ‘fed’ with new TCM containing cytokines on day 3:
Carefully remove 1ml spent medium from each well, care is required to avoid disturbing the cells.
Add 1.5ml fresh, pre-warmed TCM containing cytokines at 1:20 to each well and re-culture the DC for required culture period (typically up to 7 days).

8. At the end of the culture period adherent and non-adherent cells can be pooled for use in immunoassays and phenotyped (as required). Adherent cells may require a dissociation step to remove them from the plate.