This month's question from the Molecular Biolog y Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Cell Culture” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&A
What could cause ficoll-paque gradient centrifugation to fail? (Thread 30996)
Q Over the years, I have routinely used ficol l-paque gradient centrifugation to isolate peripheral blood mononuclear cells (PBMC) from whole blood or buffy coats. However, two weeks ago, I started experiencing difficulties with this procedure. When I lay the diluted sample on the ficoll, it immediately falls to the bottom of the tube. Then when I centrifuge as usual, I do not see the expected Erc-ficoll-PBMC-plasma layers; instead, Erc is at the bottom followed by a smooth blood gradient toward the rim of the tube.
I first thought that very low freezing temperatures might lyse my samples during transportation. I tried using a polystyrene box to keep the samples at room temperature, but this did not help. I checked all of the chemicals and they seem fine. The same student performed the separations in each case. We used the same centrifugation force. The blood samples were different each time, but this was normal. I have no idea how to address this problem. I consider the technique to be very simple, which may add to the problem since I can't think of where to troubleshoot. Can anyone offer any suggestions?
A Did your ficoll-histopaque get frozen? If so, it might have formed a density gradient, in which case you would get a different density by pipetting at different heights of the liquid.
Q I wondered about this as well, and so I made sure to pre-warm my ficoll to room temperature before using it. Unfortunately, it made no difference.
A This sounds very odd, and quite frankly, I'm not sure what is happening. But I can suggest some possibilities:
A difference in your chemicals. You say you checked them, but have you tried buying new chemicals? Try changing the ficoll and any other important reagents to different lot numbers and see if that will resolve the problem. Have the tubes you perform the centrifugation in changed?
A difference in your environment. Has anything changed in the lab? It could be something as simple as a large machine being installed nearby or someone turning up the heat.
A difference in your samples. Is the blood transported in a different way? Was there a change in the EDTA concentration? (This should not affect what you are doing too much, but it might combine with other changes to cause some difficulty.) Are the samples coming from different sources? Before adding the blood samples to the gradient, did you check to see if there were intact cells in the samples you received? To test for this, you might try removing an aliquot prior to ficoll separation. Plate this onto a matrix such as methylcult to check for colony formation and compare this with a control of fresh blood.
It seems strange to me that two out of eight of your gradients worked; this implies that either the blood source or environmental change is the cause. Other than blood, is there anything else you can try spinning through the ficoll? Try beads or dye. Then you can see if you can repeatedly get good gradients in the absence of the blood.
Q I am confident there is something trivial behind this mystery, but it's becoming a big problem. Ficoll centrifugation is very important to our cell culture experiments and now we are stuck.
A I often use ficoll separations and have found smeared gradients when there is something wrong with the brake settings of the centrifuge. Is it possible that someone adjusted your brake settings?
A Along those lines, are you sure the rotor is well balanced? Water left inside a bucket can make it imbalanced, which is like trying to centrifuge on a vortex. That would surely affect your separations.
A If it isn't the centrifuge, you might check your diluent and tubes. Could the problem stem from the solution you are using to dilute the sample? Have you tried fresh dilution solution?
Are the tubes you use for the gradient disposable? If not, could residual detergents or chemicals from other experiments still be present to disrupt the gradients?
A You mentioned that the samples begin to fall down through the gradient when you first add them to the ficoll, even before centrifugation. That's actually quite normal with the Ficoll-Hypaque I use.
Q After several weeks of testing, I figured out that the problem was just a change in how we diluted the blood samples. For years, we used commercially prepared 1× PBS, but a few weeks ago, I ordered 10× PBS to save some money, but didn't think to inform my students of the change.
Well, what we found is that if you dilute blood or buffy coat with 10× PBS, you get immediate precipitation of Erc and probably some other parts of the blood through the ficoll gradient. Just as I thought, it turned out to be something small and trivial. You never can be too careful.