Hello Diana Nelson,

Both the cellular content of the sample itself, and the volume/height of the sample on top of Ficoll are important.

The height of the sample on top of the Ficoll gradient directly influences the path length that cells must travel during centrifugation. A greater height can lead to increased red blood cell contamination in the isolated mononuclear cell layer, as red blood cells have a longer distance to sediment through the gradient. While the cellular content, specifically the concentration of leukocytes, is crucial for achieving a good yield of the desired cell population. If the sample is too dilute, the number of target cells recovered may be reduced. Conversely, an overly concentrated sample can lead to aggregation and inefficient separation.

When scaling down a Ficoll protocol, a balance must be struck between maintaining an appropriate sample height and ensuring a sufficient concentration of target cells. For instance, if the original protocol used 10ml of blood on 3ml of Ficoll, a smaller-scale protocol might involve using a reduced volume of diluted blood, such as 3ml of diluted blood, on a proportionally smaller volume of Ficoll, ensuring that the height of the sample layer is maintained within an acceptable range for efficient separation.

So, I would prefer the manufacturer’s recommendations as dilution decreases the density of the cell suspension and reduces the aggregation of RBCs, leading to a higher yield and greater purity of MNCs. The 10 ml leukocyte suspension, while rich in white blood cells, is likely too concentrated for optimal separation and could be too tall in a smaller tube.

Best,