I have been doing mononuclear cell prepartions from human cord blood samples for decades. I always dilute the starting material 1:1 with Dulbecco's PBS with 1% human serum albumin.

The problem with undiluted blood is that its' density is too close to ficoll for adequate separation of cells. Dilution of blood by 1:1 or 1:2 decreases the density of the blood sufficiently to allow separation of RBC from WBC.

When you collect the mononuclear cell interface, you will transfer some ficoll along with the cells you want, so you need to dilute that a minimum of 1:3 or the cells will not pellet properly.

Red blood cells will aggregate in Ficoll, by diluting the buffycoat less white blood cells will be in the aggregate. If number is less important you can skip the diluting of the buffycoat.

We dilute 1:1 (15ml blood + 15ml PBS w/o Ca++ and Mg++)and separate over 15ml Ficoll to achieve best separation and max yield of viable cells. Good Luck!

Then I guess loading as much buffy coat volume/ficoll tube is also beneficial, to reduce the ficoll exposure. Thanks for the answer!

As already mentioned, aggregation of erythrocytes is reduced with dilution. 1:1 is just about enough. Cell viability remains higher with dilution, and even then preferably with suitable culture medium. Other media like PBS when used should not have salt concentrations too high, forexample about 10 mM phosphate is okay.

I have been doing mononuclear cell prepartions from human cord blood samples for decades. I always dilute the starting material 1:1 with Dulbecco's PBS with 1% human serum albumin.

The problem with undiluted blood is that its' density is too close to ficoll for adequate separation of cells. Dilution of blood by 1:1 or 1:2 decreases the density of the blood sufficiently to allow separation of RBC from WBC.

When you collect the mononuclear cell interface, you will transfer some ficoll along with the cells you want, so you need to dilute that a minimum of 1:3 or the cells will not pellet properly.

Any buffer or Media (like RPMI) can be used to dilute the buffy coat. This is pretty essential as cell count of buffy coat needs to be brought to that of whole blood (which has lesser RBCs and WBCs). Out of experience, adding EDTA can prevent clumping of cells, but make sure that it will not interfere in your end use of the cells.

We dilute 1:3 of buffy coat with PBS and the yield of viable cells is high, it gives better separation. Good luck..

In our lab we routinely dilute 25 mL blood with 15 mL Dulbecco's PBS and then underlay with Ficoll. This provides adequate dilution to allow separation and minimize aggregation without unnecessarily increasing the number of tubes. If step by step video instructions would be helpful you may consult our video article in JOVE:

http://www.jove.com/index/details.stp?id=1167

You get a better banding of PBMCs if you dilute with PBS. whole blood is too dense if you load it straight. Yes you can do it without but you will loose cells and have alot of platelets in your sample. In general you should use at minumum ~ 1:4 ratio of PBS diluted blood to ficoll.

Good luck with your research

A buffy coat is a cell concentrate from blood samples, Dilution of buffy coats restores initial cell concentration

Thank you all for your answers, now I can better decide when it's necessary to dilute the buffy and when I can skip that step.

decreasing the tendency for trapping of lymphocytes in the RBC layer, prevention of clumps are the main reasons: I would suggest that these are done at 18-20deg C and not at 37 or 4 deg as they also minimise yields of mononuclear cells;

As other people alredy said, dilution of buffy coat before loading on Ficoll is important to restore initial cell concentration and avoid RBCs sticking on the PBMC layer. I prefer to use RPMI (WITHOUTH HEPES) to dilute the buffy coat. On the contrary, separation from whole blood is working very nicely, so you don't need to dilute the blood. Hope that helps.

I never dilute the buffy coat samples before ficoll separation (I use Histpaque from Sigma cat # 1077) and I do get good yield. And it is always preferable to do the centrifugation in lesser no of tubes for one sample because, the more the number of tubes, the more cells you will lose while collecting the interface cell layer after centrifugation and it also increases the serum/histopaque contamination. As for the quick loading, the blood / buffy coat should be loaded along the walls of the tube so that it forms a distinct layer over the histopaque and in each tube, the histopaque and the sample should be in 1:1 ratio. If you delay too much in the loading of the buffy coat over the histopaque, the sample will start settling even before you start spinning it and the separation will not be proper. in this case, not only you will lose the PBMCs but also might get RBC contamination in the PBMCs. So you should be fast but careful enough while loading the buffy sample such that it lies over the histopaque but does not settle down before spinning.

Dilution is important to avoid RBCs contamination, we do dilute (apx) 60ml to 200ml with PBS and always give best separartion.

This will prevent the loss of Lymphocytes which were clumped together, also make the easy separation because of homogeneous and less hindrance while centrifugation.

Yes. It is important to get a lower density before Ficoll gradient to get a nice separation of PBMCs

For better separation and avoid of Lymphocytes clumping dilution is necessary

I did it using the method of Valentina Barrera since years ! To note if you may have cord blood you have also to dilute a few to obtain better results.

Prevent aggregation, improve separation

Yes from me as well! As it has been said is crucial for a better lymphocyte yeld. I also have used RPMI w ith good results., 1: 2 . Also as Chandra w rote re m ember the temp. As well.

Good luck!