As part of the immune system, dendritic cells (DC) play a pivotal role in catalyzing the adaptive immune response. Once they capture and process local antigens, they migrate to draining lymph nodes in order to prime T lymphocytes. For their essential function, they are more and more exploited in tumor immunotherapies.
Since DC are often used in vitro screens, the need for a reliable method for their transfection became a key step. For this purpose, we try to present here a short guideline for the transfection of human DC cells (monocyte-derived) in suspension, using the 4D-NucleofectorTM Device.
In the case of immature DCs after thawing the cells you can culture them in T75 flasks at 2.5 E+05 cells per flask with the corresponding culture medium (RPMI1640 + 10% FCS + 2% Ultraglutamin + 1% Na-Pyruvat + 1% PS + 25 ng/ml IL-4 + 50 ng/ml GM-CSF) for 5 days. Change medium on day 2 and 5. For the mature phenotype, change the medium on day 5 to differentiation medium (RPMI1640 + 10% FCS + 2% Ultraglutamine + 1% Na-Pyruvat + 1% PS + 25 ng/ml IL-4 + 50 ng/ml GM-CSF + 10 ng/ml IL-1β + 20 ng/ml TNF-α + 10ng/ml IL-6 + 500 ng/ml PGE2) and incubate for 24 hours.
For the transfection using NucleofectorTM Technology, we recommend to use per sample 2 x 106 (for cuvettes) and 4 x 105 (for strips). To collect the cells, centrifuge them at 90xg for 10 minutes at room temperature. Resuspend the cell pellet carefully in room temperature nucleofection solution P3 (mixed with the supplement). Prepare mastermixes by dividing cell suspension according to number of substrates. Add required amount of substrates to each aliquot (max. 10% of final sample volume). For the DCs it is important when using mRNA as substrate to pipette it directly into the cuvette, then add the cell suspension and immediately transfect the sample. This step is recommended because the cells, once in contact with the mRNA, will start to degrade it, resulting in suboptimal transfection efficiencies.
For the transfection, we would recommend depending on the substrate either CB-150 program for mRNA or EH-104 for DNA.
In order to have a better viability after the transfection, we suggest to include a recovery step immediately after applying the pulse: add culture or differentiation medium to cuvette (500 µl) or 80 µl to each well of the strip and allow to stand at room temperature for 10 min.
Mix cells by gently pipetting up and down two to three times. Plate desired amount of cells in culture system of your choice. Immature and mature DCs should be kept in their respective medium after nucleofection until nucleofection results will be analyzed.
The maturation of immature DCs can also be started directly after transfection using NucleofectionTM Technology. In this case, differentiation medium should be added for the recovery step and the cells will be transferred into differentiation medium.
Since the results might be donor dependent, we would recommend to check the transfection efficiency at different time points and in the first experiment to use the pmaxGFP plasmid provided in the kit, as a positive control. Based on the results of the initial set of transfections, we encourage you to contact the Scientific Support Team for further optimizations.
We wish you happy transfections!
Written by Elke & Gabriel
Scientific Support Specialists, Lonza Pharma-Bioscience Solutions at Lonza