Hello,
I am looking for a protocol to differentiate dendritic cells from monocytes.
I would like to isolate monocytes from PBMCs using the EasySep™ Human Monocyte Isolation Kit (StemCell).
I found information that DC can be obtained by culturing monocytes with IL-4 and GM-CSF for several days. Various cytokine concentrations and culture times are reported in the publications. Has anyone differentiated DCs this way and has a good protocol?
Or are you using the ImmunoCult™ (StemCell) dendritic cell culture kit?
Kindly refer to the following:
doi: 10.1084/jem.180.1.83.
doi: 10.1084/jem.179.4.1109.
Hi Klaudia,
I've done it for several years now. It's pretty straightforward. In brief:
PBMCs were obtained by LymphoprepTM (density gradient from buffy coats of healthy human donors). CD14+ cells were purified using human anti-CD14-labeled magnetic beads and LS columns. Cells were plated (1 × 106 cells/mL) in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 200 mM L-Glutamine. To generate monocyte-derived dendritic cells, the medium was supplemented with 50 ng/mL of human GM-CSF and 25 ng/mL of human IL-4 at days 0 and 3; and incubated at 37 °C and 5% of CO2. At day 5-6, you can harvest immature dendritic cells (iDCs).
Or if you want mature DCs you can add at day 5 GMCSF,IL-4 plus TNFa, PGE2 and IL-1B.
Also, eluted CD14+ cells were labeled with anti-human antibody CD14-FITC to determine yield and purity, at least 96% is optimal.
You can refer at 10.1002/JLB.4MA0320-544RR.
Hope it helps.
Hi Klaudia,
My name is Sneha Balani and I am the Product Manager for Immune Cell Culture Products at STEMCELL Technologies.
I recommend using the ImmunoCult DC Culture Kit for differentiating DCs from Monocytes after isolation with EasySep. We have optimized our product to consistently provide good results while saving our customers the time of sourcing and testing individual cytokines. You can find the full protocol here: https://cdn.stemcell.com/media/files/brochure/BR27017-Dendritic_Cell_Research.pdf
I hope this helps! If you have any more questions, feel free to contact our product support specialists at [email protected].
Kind regards,
Sneha Balani
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