Begin isolating peripheral blood mononuclear cells by accessing the human buffy coat and transferring seven milliliters into a sterile 15 milliliters conical tube. Add six milliliters of PBS solution for a preliminary wash. Centrifuge the tube for 10 minutes at 1, 100 G in a centrifuge with a swing rotor, and brake off at room temperature.
Collect the leukocyte suspension using a Pasture pipette and transfer it to a new sterile 15 milliliter conical tube. Add PBS solution to the leukocyte suspension until it reaches 10 milliliters and mix it gently by pipetting up and down. Next prepare the density gradient solution by placing three milliliters of density gradient medium into a new sterile 15 milliliters conical tube.
Allow it to reach room temperature. Slowly add five milliliters of diluted leukocyte suspension on the conical tube walls containing the density gradient medium to create a five to three density gradient separation. Avoid disturbing the medium.
Proceed to gradient separation by centrifuging the suspension present in the density gradient medium using a centrifuge with a swing rotor and the brake off. Next carefully transfer up to 25 milliliters of the thin layer of PBMCs to a new 50 milliliters conical tube filled with PBS using a Pasture pipette and mix it well to remove residual cells and debris, centrifuge the samples at room temperature for 10 minutes at 600 G with a normal brake. Discard the supernatant and fill the sample to 10 milliliters using PBS.
Take an aliquot to count the cells. To remove the platelets, centrifuge them with a normal brake and carefully invert the tube to discard the supernatant. For monocyte CD14 positive isolation using magnetic activated cell sorting, resuspend the cell pellet in the microbeads buffer and CD14 immuno magnetic beads.
Incubate the cell suspension for 15 minutes at four degrees Celsius. To remove the unbound beads, add one to two milliliters of microbeads buffer per one times 10 to the seventh cells before centrifuging the suspension at room temperature for 10 minutes at 600 G.Then carefully invert the tube to discard the supernatant. Prepare the LS column and place it on the magnet before use.
Rinse it with three milliliters of microbeads buffer and immediately resuspend the cell pellet in 500 microliters of microbeads buffer per one times 10 to the eighth cells. Add the cell suspension to the LS column inlet and collect the negative cell fraction in a 15 milliliter conical tube below the column outlet. Wash the column three times with three milliliters of microbead buffer.
After the final wash, remove the column from the magnet and place it on a sterile 15 milliliter conical tube. Pipette five milliliters of microbeads buffer into the column inlet. Immediately insert the syringe plunger filled with the target cells into the column inlet and dispense the cells in the column.
Then centrifuge CD14 negative and CD14 positive cell fractions and discard the supernatant. For monocyte differentiation into human monocyte derived dendritic cells, prepare a cell suspension containing 1.3 times 10 to the sixth cells per milliliter by adding the appropriate volume of differentiation medium to the CD14 positive cells. Resuspend the monocytes by pipetting with a Pasture pipette.
After plating the 1.3 times 10 to the sixth cells per milliliter suspension per well of a 24 well plate, incubate in a culture incubator at 37 degrees Celsius with 5%carbon dioxide. Change the culture medium and supplement it with fresh cytokines every two to three days. To collect the differentiated cells, transfer the entire cell suspension to a sterile conical tube using a micro pipette and wash the culture flask twice with PBS.
After centrifuging the conical tubes at room temperature for 10 minutes at 180 G, resuspend the pellet in the appropriate media or buffer for the experimental setup. During the monocyte differentiation, interleukin four and granulocyte macrophage colony stimulating factor stimulation changed the cell phenotype. Data showed that human monocyte derived dendritic cells lost the expression of surface marker CD14, mainly expressed by monocytes and gained significant expression of CD1A.
a marker expressed by human dendritic cells. Human monocyte derived dendritic cells also obtain higher expression of MHC2 HLA-DR, an antigen presenting molecule expressed by human dendritic cells and other antigen presenting cells.
