Your ficoll is at room temperature and you centrifuge at room temperature?  The density of the gradient is temperature sensitive and it must all be performed at RT.

hello please try to use RPMI medium instead of the PBS.

also use the ACK lysis buffer to remove the red blood cells from the lymphocyte cells.

i have used the same protocol to get rid of the RBCs.

Dear all,

thank you very much these answers. I will try all of idea :)

Michel: yes everything is room temperature !

hello IIdiko 

also try to remove the plasma from blood and then add the equal volume of the RPMI medium and then overlay on the ficoll. and them wash the cells with the medium  and later was it with the ACK lysis buffer. try this 

Step 3: increase from 400 x g to 1000 x g

Every time you spin Ficoll or any gradient separation, the protocol is to use NO BRAKE in the centrifuge.  Another possibility is if you are bleeding the same donor frequently, reticulocytes (RBC precursors with nucleai - which are heavier than RBCs) tend to accumulate in the same layer with mononuclear cells. They usually do not affect tissue culture, cloning or other procedures with the MNC.

Hello Ildiko, I found that a main factor compromising PBMC purity from RBC after ficoll separation is the delay between drawing and processing. With 24h old blood, a clear separation in one step is nearly impossible, and Heparin as anticoagulant perform better than EDTA or ACD for long delays. I Hope it helps. Mario

Here are some proposals which I think may be helpful:

1- For density gradient centrifugation, you can centrifuge for 20 min without brakes at 800 × g or for 30 min at 500 xg all at room temperature...

2-When removing the buffy coat,  it is important that no Ficoll is taken along with the WBCs as this can damage the cells (Ficoll is VERY toxic to cells).

3- Plates that have been coated with cell attachment agents should be allowed to dry under the hood after these coating agents have been aspirated, before you add culture medium...

Hello, thank you for your question! A lot of my points are just repeats of what other people have already said. There are one or two things that will be different, however.

1. The Ficoll-Paque PLUS reagent should be at room temperature.

2. There really is no need to use any kind of red blood cell lysing buffer; all the RBCs should pellet. ACK = ammonium-chloride-potassium (which are the main ingredients in RBC lysing buffer)

3. Your blood sample should be diluted 1:1 in PBS or PBS with 2% FBS (this improves your yield from the blood). 

4. Centrifuge the diluted blood through the Ficoll gradient at 1200 x g with NO BRAKE (the total for the spin is about 45 minutes...not fun I know hehe)

5. If you are harvesting PBMCs (i.e. no negative selection for purified T cells), the white lymphocyte layer should be removed in one shot with a disposable transfer pipet; otherwise, the lymphocyte layer will be less dense and require more "transfers" to obtain all of the T cells.

6. It shouldn't matter if you wash with complete RPMI media, regular RPMI media, or with PBS with 2% FBS. You are doing the right thing by completely filling up the conical or tube in order to wash and dilute out any residual Ficoll, and two washes are most definitely needed.

7. I would spin at 1200 RPM for 3 MINUTES when doing the washes; I think 10 minutes is too long and can hurt your viability.

8. After this, you can resuspend the cells in whatever volume of complete RPMI media or PBS with 2% FBS depending on whether you plan to culture them, stain them for FACS, etc. If you resuspend in PBS with 2% FBS, I would keep them at 4 degrees; if you resuspend them in complete RPMI media, I would keep them at 37 degrees.

I am basing my comments on the RosetteSep protocol from StemCell Technologies. I was routinely using this product (#15061 if you want to look up their protocol) to isolate purified T cells from LeukoPaks. If you just want PBMCs instead of purified T cells, you, of course, just don't use the negative selection cocktail. They also have a nice table for what volume of Ficoll to use depending on the volume of your diluted blood sample (blood + PBS).

I hope this helps! Good luck!

Dear all :)

Thank you very very much a lot of help and advice.

I would try everything !

I have got one more question:

I will use lymphocytes for EliSpot analysis. And I use a commercial kit. The kit says me, I have to use AIM V medium (serum free medium) instead of RPMI. Because the T lymphocytes can stimulated faster in RPMI reagent.

If I use PBS+2% FBS, will not it stimulation?

Thank you :)

It's best to use the donor own plasma that you're saving from the ficoll. If you dilute your heparinized blood with the same volume of PBS before the gradient (brakes off in the centrifuge) your plasma will be 50%. You can use 5% of this plasma to keep your mononuclear cells with antigen during stimulation.  All the details are in this old publication: PubMed PMID: 10825580. Good luck!

Two points:

1. As suggested by many - increase the spin time or g force.

2. Also, I'd check your anticoagulant.

On a side note, Can you fix a bad overlay? 50mL conical tube ficoll gradient if when you are overlaying the blood the pipette gun seal breaks so the blood is homogenized instead of making a layer? salvagable? not salvagable? Right now my mistake tube is essentially ballast ;-(

After talking to tech support for my question. You can re-ficoll. Its not ideal and repeat yields and quality will be lower. But, you can try and salvage the top of rbc layer, or the entire rbc part if necessary and re-ficoll overlay. basically, spin the bad tube, look at it and if you think you have too, salvage where you think the pbmcs are and re-ficoll that. Check them before final pooling with the good tube though.