Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes
Transcription
Seed four sets of CD14+ monocytes in a concentration of 0.3 to 0.5 x 106 per milliliter of cell culture medium of IL-4 in six-well plates. Incubate the cells in a tissue culture incubator at 37 degrees Celsius with 5% CO2. On day 4, remove 850 microliters of medium from the culture, and centrifuge.
Aspirate the supernatant, and resuspend the pellet in 1 milliliter of cell culture medium. Add the cell mixture back to the culture. On day 5, add 1 microliter of vitamin D3 stock and 1 microliter of dexamethasone stock per milliliter of medium to two of the sets to generate tolerogenic moDCs.
On day 6, add 200 nanograms per milliliter of GM-CSF and 200 nanograms per milliliter of IL-4 to all of the sets. Add 1 microgram per milliliter of LPS to one of the sets treated with only GM-CSF and IL-4 to generate mature moDCs.
Add 1 microgram per milliliter of LPS to one set of the tolerogenic modes moDCs to generate LPS-tolerogenic moDCs. Finally, on day 7, harvest the different types of moDCs by flushing the culture dish with PBS-EDTA.
