Bu içeriği görüntülemek için JoVE aboneliği gereklidir.  Oturum açın veya ücretsiz deneme sürümünü başlatın.
Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

DEŞİFRE METNİ

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.

İlgili Videolar

Read Article