Hello all,
I present here a problem to see if someone has encountered it and can provide some clarity. We are trying to isolate neutrophils from patient blood in tubes EDTA. We are following the protocol in
Article Protocol for density gradient neutrophil isolation and flow ...
which is quite straightforward. We have a lot of experience using Percoll and Ficoll solutions, so I am positive it is not a matter of bad layering, wrong calculations when preparing the solution, wrong centrifuge settings (acc-brake, etc.)
According to the protocol, there are 2 centrifugations, which should result in a separation such as:
-Serum
-Lymphos
-Percoll
-Neutros
-Percoll
-RBC
After the first centrifugation (200g), the blood trespassed the 62% layer, staying in the middle of the tube. However, after the second centrifugation (400g), nothing happened, the blood was unable to trespass the second layer. Moreover, we started to increase the speed of these centrifugations and went up to 1000g (in for a penny...), and still nothing happened. The blood was unable to trespass the second layer, and I am quite puzzled, since 1000g is a very high force and you would expect most cell types to pellet. See attached.
There was a fine cloud on top of the blood and after going to the cytometer we could see all immune cell types, meaning no immune population was isolated.
What could have gone wrong? I would understand bad separation, low yield, etc. But a whole 15ml of blood not depositing, even a 1000g?
Thank you in advance.
Hey Sergio,
I have never done a Ficoll like that. So I am just speculating about the chemistry. Is it possible that the EDTA in your sample and the ions in the PBS used for dilution are creating some kind of chelate effect? The Ficoll in your picture is looking a little bit cloudy. Is the Ficoll layer solid or gel like?
Best
Kevin
Hi Kevin,
It is Percoll, not Ficoll (very similar).
They are all liquid phases, with different density. The blood one (diluted with media), then the 62% Percoll (sacarose solution), then the 75% Percoll. They are rather dense solutions but not solid or "gel-ish" at all.
About the chelate effect, I am not well versed in chemistry, but in the past, when isolating lymphocytes, we always did K3 EDTA and PBS and no strange phenomonon occurred, to my knowledge
The rbc are not forming a layer at the bottom of the tube which suggests that the percoll is too dense so may not be dituted to the correct percentage of percoll, This may be an area to check that all reagents are at the correct dilution
- Badges
- Science topic
- Similar topics
- Medicine
- Physiology
- Biosignals
- Biomedical Signal Processing
- Statistical Analysis