Dendritic cells do not divide, but they can be cultured and activated. Keep these tips in mind.
- Medium: We use X-VIVO™ 15 or X-VIVO™ 20. If you prefer serum-containing medium, RPMI 1640 supplemented with 10% fetal calf serum is suggested.
- Culture Flasks or Dishes
- Thaw and count cells following standard procedure.
- Adjust cell concentration to 500,000 cells per mL. This concentration can be adjusted based on your protocols.
- Add cells to culture at 100,000–200,000 cells per cm².
- Cells can be used for experiments immediately or after overnight culture.
Collection of Cells from Culture
- Collect medium from the culture and place in a tube. Immediately add PBS to the adherent cells. Pipet up and down to rinse the culture surface and pool this with the cell in medium.
- Add PBS to the culture and place the culture in the incubator at 37°C for 10–15 minutes. During this time, the cells should begin to detach from the culture surface. They become rounder and brighter when observed using a phase contrast microscope.
- If cells are in a flask, the flask can be agitated and rapped against the heel of your hand to dislodge the cells. If using culture dishes or wells, pipet the PBS forcefully against the culture surface to dislodge the cells.
- Collect the cells in PBS and pool with the previously collected cells.
- DC grow as a mixture of adherent and non-adherent cells with the proportion of adherent cells dependent on the culture medium and activation stimuli (if added). This complicates moving them from one culture vessel to the next or performing experiments that involve collecting intact cells. Recovery of the cells is always lower than the starting cell number. We recommend setting up the cells in the culture configuration you will use for your experiment. If transfer out of the culture vessel is necessary, use the procedure outlined above.
- Dendritic cells do not proliferate. They will survive in culture for 1 week or more. If cells are to be maintained for more than overnight culture, addition of 500 U/mL GM-CSF and 500 U/mL IL4 is recommended to ensure maintenance of DC phenotype.
- Dendritic cells from Cellero have not been activated. They are considered immature as measured by expression of CD83.
- DC can be stimulated with LPS, but serum must be included. Serum provides soluble CD14, which binds the LPS and facilitates binding to TLR4. Fetal bovine serum can be used.