We are again describing a rare and very simple protocol here:
The applications for human mononuclear cells (MNCs) are increasing, as these cells can be used for various tests, cell therapies and the development of human therapeutic antibodies. Density gradient isolation is still used today to separate MNCs. Diluted blood or buffy coat is added to the density gradient liquid, which is then centrifuged to obtain the different fractions. The white layer contains the MNCs, which are carefully pipetted out and washed to obtain the cells. The disadvantage of this laborious procedure is that blood and density gradient fluid may mix, which can lead to wastage of the valuable blood sample.
We have developed a simple procedure in which the user waits a few minutes after mixing the blood sample in the Quick MNC isolator in a tube. The sample is then washed two or three times with PBS and centrifuged to obtain a pellet. Although small amounts of red blood cells occasionally remain in the pellet, they have no effect on the cells in the assay, e.g. when separating with magnetic beads, or on further experiments with isolated MNC cells. A high cell yield can be achieved with a single milliliter of blood or buffy coat. This MNC isolator method is an extremely fast, simple and effective method. Even very small amounts of buffy coat or blood, e.g. 100µl, can be used to isolate MNC.
There is no documented case study in the literature using the density gradient method to separate MNC from such small amounts of blood or buffy coat. Our method is only option for small volumes.
If the user cultivates the MNC in media with fetal calf serum (FCS), it is sufficient to use 2-3% FCS instead of the usual 10% FCS. This helps to reduce the financial burden on laboratories. Please read our publication in the reference section.
Using this protocol, we have developed many cell cultures with MNC over the last 14 years.