I carried out PBMC extraction using Ficoll paque PLUS. However after using PBS washed for twice, cells clump together and cannot be resuspended. Could anyone help to see what the problem is?
Pic of cell clumps are here (sorry not so clear):
http://imgur.com/wzvhGLK
http://imgur.com/h898A5h
The protocol used are as follows:
Blood from healthy person used was collected in EDTA tube. Fresh blood collected 2-3 hrs before used. Store at room temp.
Before used, blood was diluted by PBS (room temp, pH=7.4, no Ca++ and Mg++, no EDTA or other anticoagulant added, http://www.lifetechnologies.com/order/catalog/product/21600010?CID=search-21600010)
12ml blood diluted with 12 ml PBS, and the 24 ml diluted blood layer on 15ml Ficoll (room temp). Centrifuge for 20 mins, no brake, 2200rpm
Then get the PBMC layer and put it in 15ml tube.
A little bit of plasma and Ficoll are also get together with PBMC (around 2.5 – 3ml), then fill the tube with PBS (same solution used as above). 1800rpm 10mins
After the first wash the cells can still be resuspended
Wash one more time using PBS. 1800rpm 10 mins.
However, after this wash the cell clumped together and cannot be resuspended (I suppose they are cells…)
I have performed two times of PBMC extraction using two different healthy people’s blood but cells clump together in all two trial.
I would like to ask:
why cells clumped together after my second wash?
How can I prevent the cells clumped together?
- Too much blood used and therefore too much cells in one tube?
- Problem of PBS?
- get too much plasma/Ficoll?
- temp of the solution used?
- my technique problem?
Can anyone please help me how to improve the problem?
Protocol seems normal for exception several notes. You need to add more PBS to dilute whole blood (24 ml vs 12). Using rpm is not good to describe centrifugation conditions, because diameters of rotors may be quite different. Use G. Centrifugation on Ficoll gradient should be performed at 400G, 20-40 min. Washings of MNC should be at 200 G, 10 min. Obviously there are two reasons for clamping: 1) Increased number of platelets in collected MNC (in this case you can decrease the clamping by the use of heparinized tubes instead of EDTA tubes for blood collection) or 2) cell death due to mechanical or chemical damage (check PBS, rpm, add 5% FBS or heat-inactivated IV(AB) serum into solution for washing of MNC).
Thanks Bryan and Dmitry.
I will check the G force tomorrow morning when I go back lab.
But the 2200/1800 rpm I used is actually "borrow" from a group that separate PBMC from buffy coat almost daily, using the same centrifuge as we are using the same facility. (The buffy coat they used was not fresh, at least collected one day before use and stored in 4oC)
So maybe because fresh cells are more easily being activated by mechanical force?
And one more thing I almost forget to tell -
In the second trial, after the second wash, I remove the big clump of cells (the tube remains turbid , wash and centrifuge one more time. This time there's no big clumps formed, although there's a few much smaller clumps that could still be visualized.
I wonder if the cells are activated/dead because of the mechanical force, there would be more and more clumps formed in the third wash?
For the rpm I used previously, 2200 rpm = 974 x g; 1800 rpm = 652 x g
Toady I perform PBMC extraction using lower g force
Ficoll; 394 x g, 30 mins
Wash: 243 x g, 10 mins, wash twice
Everything is the same as above expect centrifugation
But then after the first wash, quite a lot of cells remain un-pallet as can be seen from the PBS that it's still a bit turbid.
After the second wash I count the cells, I can only extract 7-8 million cells from 12ml blood..Normally one can extract ~15 million cells from 12ml blood
What should I do to increase the cell number?
Your method looks ok, but the wash spins seem a little high from protocols I've used. You might try around 300-400 x g for the washes and see if this prevents clumping. Also, is your cell pellet clear? Sometimes you can get some RBC carry-over which will cause clumping. If you use a Pasteur or 1ml pipette to carefully remove your buffy coat you can minimize RBC, Ficoll, or serum carry-over which should help prevent clumping as well.
20min at 2000rpm without break is ok. 1800rpm for first wash is also ok but you should reduce to 1500 or 1300 rpm later.
Clumps can means cell activation or apoptosis. Your blood being fresh, I would suppose apoptosis is minor. You can improve using EDTA (2.5mM) in your dilution buffer and then use PBS+2.5mM EDTA+1%FCS as a general wash buffer. It avoid cell aggregation and help survival.
I agree with Dmitry and Sebastien I suggest you to dilute the whole blood 1:3 with PBS and for washing step it would be useful to add protein as FBS 5% or human albumin 2%.. In case of cell clumping PBS EDTA is better than PBS to avoid cell aggregation. I always use PBS/EDTA to avoid cell aggrgation in flow cytometry analysis
Hi,
In my experience, cells in buffer without protein is prone to clumping. I run the flow core at my institution and when people have clumping problems when trying to run samples on our instruments, they are almost always in PBS without FBS or BSA. And once the cells clump, you're not going to get them back into suspension by adding protein to the mix. So, you can't do one or two washes in PBS, then in buffer with protein. Have it in there at every step.
Good Luck,
Mike
Hi,
My suggestions are to dilute the blood to 24ml either with PBS or with HBSS without Ca++ and Mg++ and 20ml Ficoll. Then, transfer the PBMC layer into a 50ml tube instead of a 15ml tube and fill it with PBS or HBSS. The centrifucations are fine a part from the last one, which I normally carry out at a lower speed (i.e. 800rpm for 10min). Remove the supernatant carefully as this pellet is not very firm.
Good luck!
Storing blood (your starting material) at room temperature for that much time may in anyway impair the isolation process and PBMCs yield. Clumps are formed in our lab too, we try to prevent their formation by resuspeding first of all slowly and with a small amount of solution, and then we gradually add more of it. Simple but important steps..!
I also got the same issue while PBMC isolation but it all resolved by washing the buffy layer with PBS containing 2% FBS and low speed centrifugation.
It may help to separate the pellet to separate and re-suspend the cells by simply scraping the bottom of the conical tube across a fume hood grate. This breaks up the pellet via vibration. You could then add further media.
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