The overall goal of this protocol is to generate tolerogenic and mature dendritic cells from monocytes for experimentation or vaccine development. This method can help answer key questions in the field of immunology by providing a model to study the development, maturation and antigen presentation of dendritic cells. The main advantage of this technique is that it enables the generation of high numbers of dendritic cells in vitro for experimentation.
Begin monocyte enrichment by mixing 20 microliters of previously prepared peripheral blood mononucleal cell, or PBMC cell suspension with 20 microliters of tripan blue, to count the the number of live cells using a cytometer. Then, centrifuge the cell suspension. Aspirate the supernatant completely, and re-suspend the cell pellet to a concentration of 80 microliters of staining buffer for 10 to the seventh cells.
Add 20 microliters of CD-4 micro beads per 10 to the seventh cells. Mix well and incubate the cells for 15 minutes at four degrees Celsius. Wash the cells with one milliliter of staining buffer per 10 to the seventh cells, and centrifuge the cells.
Aspirate the supernatant completely, and re-suspend the cell pellet. Obtain a medium column for a maximum of two times 10 to the eighth total cells, and place the column in the magnetic field of the separator. Next, rinse the column with 500 microliters of staining buffer.
Pipette the cell suspension onto the column, and collect the unlabeled cells that pass through the column in a 15-milliliter tube. Replace a new 15-milliliter tube under the column and wash the column three times with 500 microliters of staining buffer. Make sure the column reservoir is empty before adding new staining buffer between washes.
Remove the column from the separator, and place it on a fresh 15-milliliter tube. Pipette one milliliter of staining buffer onto the column, and immediately flush out the magnetically labelled cells by firmly pushing the plunger into the column. Then, repeat the magnetic separation steps using the illuded fraction in a new column to increase the purity of the CD14 plus cells.
Seed four sets of CD14 plus monocytes in a concentration of zero point three to zero point five times 10 to the sixth 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 five percent CO2. On day four, remove 850 microliters of medium from the culture, and centrifuge.
Aspirate the supernatant and re-suspend the pellet in one milliliter of cell culture medium. Add the cell mixture back to the culture. On day five, add one microliter of vitamin D3 stock and one microliter of dexamethazone stock per one liter of medium to two of the sets, to generate tolerogenic moDCs.
On day six, add 200 nanograms per milliliter of GMCSF and 200 nanograms per milliliter of IL-4 to all of the sets. Add one microgram per milliliter of LPS to one of the sets treated with only GMCSF and IL-4 to generate mature moDCs. Add one microgram per milliliter of LPS to one set of the tolerogenic moDCs to generate LPS tolerogenic moDCs.
Finally, on day seven, harvest the different types of moDCs by flushing the culture dish with PVS EDTA. The immature moDCs were generated by adding GMCSF and IL-4 to purified CD-14 plus monocytes on days zero, four and six. Addition of vitamin D3 and dexamethazone post-GMCF and IL-4 resulted in the differentiation of immature moDCs into tolerogenic moDCs.
LPS was added to induce the maturation of immature moDCs to mature moDCs. And tolerogenic moDCs were stimulated with LPS to verify resistance to maturation. An analysis of DC surface markers revealed that mature moDCs express the highest levels of maturation markers HLA-DR, CD80, CD83 and CD86.
Conversely, tolerogenic moDCs displayed increased expression of CD14, BDCA3 and immunoglobulin-like transcripts compared to immature and mature moDCs. Using red CMXRos to reflect mytocondrial activity, tolerogenic moDCs were observed to have higher mytocondrial activity compared with the other moDC differentiate subtypes. After its development, this technique paved the way for researchers in the field of immunology to explore metabolic features in dendritic cells for the development of vaccines and immunotherapy.
