Hello Esraa Mosaad Soliman

1. Not diluted the blood and adding equal volume to the Ficoll separation medium.

It would not give a good yield of PBMCs.

When erythrocytes in whole blood are aggregated, some mononuclear cells are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap mononuclear cells is reduced when you dilute blood with PBS. Dilution gives a better yield of mononuclear cells and reduces the size of the red cell clumps. The more diluted the blood sample, the better the purity of the mononuclear cells.

2. Diluted the blood with PBS and added different volumes of Ficoll.

As per the standard procedure the heights of Ficoll and blood sample must be maintained. So, for 4 mL Ficoll, use 6 to 10 mL diluted blood in a 15 mL conical tube, or for larger volumes of blood to be processed with the same efficiency of separation, use centrifuge tubes of increased diameter (use 20 mL Ficoll for 30 to 35 mL diluted blood in a 50 mL conical tube) while maintaining approximately the same heights of Ficoll and blood sample. Any deviation from this may lead to contamination. For instance, increasing the height of blood sample in the tube will increase red cell contamination. As you know that the yield and degree of purity of mononuclear cells will depend to a considerable extent on the efficiency of red cell removal.

Best.

@Malcolm Nobre

So the volume of ficoll to diluted blood is according to width only of the falcon or there is a standard ratio?

And what if i diluted the blood and then added equal volume from the ficoll?.

Thanks in advance.

What method do you use for separation? Density gradient centrifugation? If you are using density gradient centrifugation, not diluting the blood will result in a less pure separation because there are so many complex components in the blood. Without diluting the blood, there is not much difference in the density of cells in the blood.

Yishen Tian Yes i will use Ficoll, after diluting the blood what volume of ficoll should i added , Equal to diluted blood or equal to the blood before dilution?